Anti-CD40 antibodies and methods of inhibiting proliferation of CD40 expressing cells

ABSTRACT

The present invention describes humanized antibodies that target CD40, wherein the antibodies comprise at least one modification relative to a parent antibody, wherein the modification alters affinity to an FcγR or alters effector function as compared to the parent antibody. Also disclosed are methods of using the antibodies of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is the national stage application of PCT PatentApplication No. PCT/US2009/031585, filed Jan. 21, 2009, and entitled“Optimized CD40 Antibodies and Methods of Using the Same,” which claimspriority to U.S. provisional application No. 61/062,172, filed on Jan.23, 2008, and entitled “Optimized CD40 Antibodies and Methods of Usingthe Same,” the contents of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to optimized proteins that target CD40,and their application, particularly for therapeutic purposes.

BACKGROUND

B Cells:

B cells are lymphocytes that play a large role in the humoral immuneresponse. They are produced in the bone marrow of most mammals, andrepresent 5-15% of the circulating lymphoid pool. The principal functionof B cells is to make antibodies against various antigens, and are anessential component of the adaptive immune system.

The human body makes millions of different types of B cells each daythat circulate in the blood and lymph performing the role of immunesurveillence. B cells, also referred to as B lymphocytes, do not produceantibodies until they become fully activated. Each B cell has a uniquereceptor protein (referred to as the B cell receptor (BCR)) on itssurface that will bind to one particular antigen. The BCR is amembrane-bound immunoglobulin, and it is this molecule that allows thedistinction of B cells from other types of lymphocytes, as well as beingthe main receptor involved in B-cell activation. Once a B cellencounters its cognate antigen and receives an additional signal from aT helper cell, it can further differentiate into the various types of Bcells listed below. The B cell may either become one of these cell typesdirectly or it may undergo an intermediate differentiation step, thegerminal center reaction, where the B cell will hypermutate the variableregion of its immunoglobulin gene and possibly undergo class switching.

B-cell development occurs through several stages, each stagerepresenting a change in the genome content at the antibody loci. Thestages of B-cell development include Progenitor B cells, Early Pro-Bcells, Late Pro-B cells, Large Pre-B cells, Small Pre-B cells, ImmatureB cells, and Mature B cells.

Mature B Cells can be Divided into Four Major Types:

B-1 cells express CD5, a marker usually found on T cells. B-1 cells alsoexpress IgM in greater quantities than IgG. They secrete natural lowaffinity polyreactive antibodies found in the serum and often havespecificities directed toward self-antigens, and common bacterialpolysaccharides. B-1 cells are present in low numbers in the lymph nodesand spleen and are instead found predominantly in the peritoneal andpleural cavities.

B-2 cells are the conventional B cells to which most texts refer. Theyreside in bone marrow, spleen, and lymph nodes. They are short-lived,and when triggered by antigens may differentiate into IgG-producingmemory B cells. In the course of these antibody responses IgG mayundergo substantial affinity maturation.

Plasma B cells (also known as plasma cells) are large B cells that havebeen exposed to antigen and produce and secrete large amounts ofantibodies, which assist in the destruction of microbes by binding andfacilitating targeting by phagocytes, as well as activation of thecomplement system. Plasma cells are sometimes referred to as antibodyfactories.

Memory B cells are formed from activated B cells that are specific tothe antigen encountered during the primary immune response. These cellslive for a long time, and can respond quickly following a secondexposure to the same antigen.

When a B cell fails in any step of the maturation process, it will dieby a mechanism called apoptosis. If it recognizes self-antigen duringthe maturation process, the B cell will become suppressed (known asanergy) or undergo apoptosis. B cells are continuously produced in thebone marrow, but only a small portion of newly made B cells survive toparticipate in the long-lived peripheral B-cell pool.

In recent years, data have emerged suggesting that B lymphocytes play abroader role in immune responses and are not merely the passiverecipients of signals that result in differentiation intoantibody-producing plasma cells. Along with their traditional roles asantigen presenting cells and precursors of antibody-producing plasmacells, B cells have also been found to regulate antigen presenting cells(APCs) and T-cell functions, produce cytokines, and expressreceptor/ligand pairs that previously had been thought to be restrictedto other cell types. Described herein are novel antibodies that havebeen optimized against B cells and methods of using them.

SUMMARY OF THE INVENTION

The present invention is directed to antibodies and methods of using thesame. In certain aspects, the antibodies include a variant Fc region. Infurther embodiments, the antibodies are humanized. The present inventionis further directed to methods of using the antibodies in variousdisease indications.

In one aspect, the present invention is directed to an antibody thatbinds CD40, wherein said antibody comprises at least one modification inthe constant region relative to a parent antibody. In one embodiment,the antibody of the invention binds with altered affinity to an Fcreceptor or alters effector function as compared to the parent antibody.

In certain aspects, the modification is an amino acid. The modificationcan be at a position selected from the group consisting of 221, 222,223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278,280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, and 337, wherein numbering is according to the EU index. The aminoacid modification can be a substitution selected from the groupconsisting of 221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E,225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E,230G, 230Y, 231E, 231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A,233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S,233T, 233V, 233W, 233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K,234M, 234N, 234P, 234Q, 234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D,235E, 235F, 235G, 235H, 235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S,235T, 235V, 235W, 235Y, 236A, 236D, 236E, 236F, 236H, 236I, 236K, 236L,236M, 236N, 236P, 236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E,237F, 237H, 237I, 237K, 237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T,237V, 237W, 237Y, 238D, 238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M,238N, 238Q, 238R, 238S, 238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G,239H, 239I, 239K, 239L, 239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W,239Y, 240A, 240I, 240M, 240T, 241D, 241E, 241L, 241R, 241S, 241W, 241Y,243E, 243H, 243L, 243Q, 243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H,246Y, 247G, 247V, 249H, 249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D,260E, 260H, 260Y, 262A, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T,264A, 264D, 264E, 264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N, 264P,264Q, 264R, 264S, 264T, 264W, 264Y, 265F, 265G, 265H, 265I, 265K, 265L,265M, 265N, 265P, 265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I,266M, 266T, 267D, 267E, 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P,267Q, 267R, 267T, 267V, 267W, 267Y, 268D, 268E, 268F, 268G, 268I, 268K,268L, 268M, 268P, 268Q, 268R, 268T, 268V, 268W, 269F, 269G, 269H, 269I,269K, 269L, 269M, 269N, 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F,270G, 270H, 270I, 270L, 270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y,271A, 271D, 271E, 271F, 271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q,271R, 271S, 271T, 271V, 271W, 271Y, 272D, 272F, 272G, 272H, 272I, 272K,272L, 272M, 272P, 272R, 272S, 272T, 272V, 272W, 272Y, 273I, 274D, 274E,274F, 274G, 274H, 274I, 274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W,274Y, 275L, 275W, 276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P,276R, 276S, 276T, 276V, 276W, 276Y, 278D, 278E, 278G, 278H, 278I, 278K,278L, 278M, 278N, 278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K,280L, 280P, 280W, 281D, 281E, 281K, 281N, 281P, 281Q, 281Y, 282E, 282G,282K, 282P, 282Y, 283G, 283H, 283K, 283L, 283P, 283R, 283Y, 284D, 284E,284L, 284N, 284Q, 284T, 284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E,286G, 286P, 286Y, 288D, 288E, 288Y, 290D, 290H, 290L, 290N, 290W, 291D,291E, 291G, 291H, 291I, 291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G,293H, 293I, 293L, 293M, 293N, 293P, 293R, 293S, 293T, 293V, 293W, 293Y,294F, 294G, 294H, 294I, 294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V,294W, 294Y, 295D, 295E, 295F, 295G, 295H, 295I, 295M, 295N, 295P, 295R,295S, 295T, 295V, 295W, 295Y, 296A, 296D, 296E, 296G, 296H, 296I, 296K,296L, 296M, 296N, 296Q, 296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G,297H, 297I, 297K, 297L, 297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W,297Y, 298A, 298D, 298E, 298F, 298H, 298I, 298K, 298M, 298N, 298Q, 298R,298T, 298W, 298Y, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L,299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E,300G, 300H, 300K, 300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W,301D, 301E, 301H, 301Y, 302I, 303D, 303E, 303Y, 304D, 304H, 304L, 304N,304T, 305E, 305T, 305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y,320D, 320F, 320G, 320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W,320Y, 322D, 322F, 322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y,323I, 324D, 324F, 324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V,324W, 324Y, 325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M,325P, 325Q, 325R, 325S, 325T, 325V, 325W, 325Y, 326E, 326I, 326L, 326P,326T, 327D, 327E, 327F, 327H, 327I, 327K, 327L, 327M, 327N, 327P, 327R,327S, 327T, 327V, 327W, 327Y, 328A, 328D, 328E, 328F, 328G, 328H, 328I,328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D,329E, 329F, 329G, 329H, 329I, 329K, 329L, 329M, 329N, 329Q, 329R, 329S,329T, 329V, 329W, 329Y, 330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N,330P, 330R, 330S, 330T, 330V, 330W, 330Y, 331D, 331F, 331H, 331I, 331L,331M, 331Q, 331R, 331T, 331V, 331W, 331Y, 332A, 332D, 332E, 332F, 332H,332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y,333A, 333F, 333H, 333I, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 334I,334L, 334P, 334T, 335D, 335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P,335R, 335S, 335V, 335W, 335Y, 336E, 336K, 336Y, 337E, 337H, and 337N,wherein numbering is according to the EU index.

In further aspects, the amino acid modification can be at a positionselected from the group consisting of 221, 222, 223, 224, 225, 227, 228,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 245,246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286,288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 313, 317, 318, 320, 322, 324, 325, 326, 327, 328, 329,330, 331, 332, 333, 334, 335, 336, and 337. In additional aspects, thesubstitution can be selected from the group consisting of 221K, 222Y,223E, 223K, 224E, 224Y, 225E, 225W, 227E, 227G, 227K, 227Y, 228E, 228G,228K, 228Y, 230A, 230E, 230G, 230Y, 231E, 231G, 231K, 231P, 231Y, 232E,232G, 232K, 232Y, 233A, 233F, 233H, 233I, 233K, 233L, 233M, 233N, 233Q,233R, 233S, 233T, 233V, 233W, 233Y, 234D, 234E, 234F, 234G, 234H, 234I,234K, 234M, 234N, 234P, 234Q, 234R, 234S, 234T, 234W, 234Y, 235D, 235F,235G, 235H, 235I, 235K, 235M, 235N, 235Q, 235R, 235S, 235T, 235V, 235W,235Y, 236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P, 236Q,236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I, 237K,237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y, 238D,238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R, 238S,238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K, 239L,239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240M, 240T, 241D,241E, 241R, 241S, 241W, 241Y, 243E, 243H, 243Q, 243R, 243W, 243Y, 245A,246D, 246H, 246Y, 247G, 247V, 249H, 249Q, 249Y, 255E, 255Y, 258H, 258S,258Y, 260D, 260E, 260H, 260Y, 262A, 262E, 262F, 262I, 262T, 263A, 263I,263M, 263T, 264D, 264E, 264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N,264P, 264Q, 264R, 264S, 264T, 264W, 264Y, 265F, 265G, 265H, 265I, 265K,265L, 265M, 265P, 265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I,266M, 266T, 267D, 267E, 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P,267Q, 267R, 267V, 267W, 267Y, 268F, 268G, 268I, 268M, 268P, 268T, 268V,268W, 269F, 269G, 269H, 269I, 269L, 269M, 269N, 269P, 269R, 269S, 269T,269V, 269W, 269Y, 270F, 270G, 270H, 270I, 270L, 270M, 270P, 270Q, 270R,270S, 270T, 270W, 270Y, 271A, 271D, 271E, 271F, 271G, 271H, 271I, 271K,271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 272F, 272G,272H, 272I, 272K, 272L, 272M, 272P, 272R, 272S, 272T, 272V, 272W, 272Y,274D, 274E, 274F, 274G, 274H, 274I, 274L, 274M, 274P, 274R, 274T, 274V,274W, 274Y, 275W, 276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P,276R, 276S, 276T, 276V, 276W, 278D, 278E, 278G, 278H, 278I, 278K, 278L,278M, 278N, 278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280P, 280W,281E, 281K, 281N, 281P, 281Y, 282G, 282P, 282Y, 283G, 283H, 283K, 283L,283P, 283R, 283Y, 284L, 284N, 284Q, 284T, 284Y, 285K, 285Q, 285W, 285Y,286G, 286P, 286Y, 288Y, 290H, 290L, 290W, 291D, 291E, 291G, 291H, 291I,291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L, 293M,293N, 293P, 293R, 293S, 293T, 293W, 293Y, 294F, 294G, 294H, 294I, 294K,294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D, 295F, 295G,295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V, 295W, 295Y, 296A,296D, 296E, 296G, 296I, 296K, 296L, 296M, 296N, 296Q, 296R, 296S, 296T,296V, 297D, 297E, 297F, 297G, 297H, 291I, 297K, 297L, 297M, 297P, 297R,297S, 297T, 297V, 297W, 297Y, 298E, 298F, 298H, 298I, 298K, 298M, 298Q,298R, 298W, 298Y, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L,299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E,300G, 300H, 300K, 300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W,301D, 301E, 301Y, 302I, 303D, 303E, 303Y, 304H, 304L, 304N, 304T, 305E,305T, 305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F,320G, 320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D,322F, 322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y, 324D, 324F,324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A,325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R,325S, 325T, 325V, 325W, 325Y, 326L, 326P, 326T, 327D, 327E, 327F, 327H,327I, 327K, 327L, 327M, 327P, 327R, 327V, 327W, 327Y, 328A, 328D, 328E,328F, 328G, 328H, 328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T, 328V,328W, 328Y, 329D, 329E, 329F, 329G, 329H, 329I, 329K, 329L, 329M, 329N,329Q, 329R, 329S, 329T, 329V, 329W, 329Y, 330E, 330F, 330H, 330I, 330L,330M, 330N, 330P, 330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q,331R, 331T, 331V, 331W, 331Y, 332A, 332F, 332H, 332L, 332M, 332N, 332P,332Q, 332S, 332T, 332V, 332W, 332Y, 333F, 333H, 333I, 333L, 333M, 333P,333T, 333Y, 334F, 334P, 334T, 335D, 335F, 335G, 335H, 335I, 335L, 335M,335P, 335R, 335S, 335V, 335W, 335Y, 336E, 336K, 336Y, 337H, and 337N.

In further aspects, the modification is at a position selected from thegroup consisting of 221, 222, 223, 224, 225, 228, 230, 231, 232, 240,244, 245, 247, 262, 263, 266, 271, 273, 275, 281, 284, 291, 299, 302,304, 313, 323, 325, 328, 332, 336, wherein the positional numbering isaccording to the EU index. In additional aspects, the modification isselected from the group consisting of 221K, 221Y, 222E, 222Y, 223E,223K, 224E, 224Y, 225E, 225K, 225W, 228E, 228G, 228K, 228Y, 230A, 230E,230G, 230Y, 231E, 231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 240A,240I, 240M, 240T, 244H, 245A, 247G, 247V, 262A, 262E, 262F, 262I, 262T,263A, 263I, 263M, 263T, 266A, 266I, 266M, 266T, 271A, 271D, 271E, 271F,271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V,271W, 271Y, 273I, 275L, 275W, 281D, 281E, 281K, 281N, 281P, 281Q, 281Y,284D, 284E, 284L, 284N, 284Q, 284T, 284Y, 291D, 291E, 291G, 291H, 291I,291Q, 291T, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M,299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 304D, 304H, 304L, 304N,304T, 313F, 323I, 325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L,325M, 325P, 325Q, 325R, 325S, 325T, 325V, 325W, 325Y, 328A, 328D, 328E,328F, 328G, 328H, 328I, 328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T,328V, 328W, 328Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M, 332N,332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 336E, 336K, and 336Y.

The antibody can further include a second amino acid modification at aposition selected from the group consisting of 221, 222, 223, 224, 225,227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 282,283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337,wherein numbering is according to the EU index. The second amino acidmodification can be a substitution selected from the group consisting of221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E,227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E,231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G,233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W,233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P,234Q, 234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F, 235G,235H, 235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W,235Y, 236A, 236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P,236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I,237K, 237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y,238D, 238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R,238S, 238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K,239L, 239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240A, 240I,240M, 240T, 241D, 241E, 241L, 241R, 241S, 241W, 241Y, 243E, 243H, 243L,243Q, 243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V,249H, 249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 260H, 260Y,262A, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 264A, 264D, 264E,264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N, 264P, 264Q, 264R, 264S,264T, 264W, 264Y, 265F, 265G, 265H, 265I, 265K, 265L, 265M, 265N, 265P,265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I, 266M, 266T, 267D,267E, 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P, 267Q, 267R, 267T,267V, 267W, 267Y, 268D, 268E, 268F, 268G, 268I, 268K, 268L, 268M, 268P,268Q, 268R, 268T, 268V, 268W, 269F, 269G, 269H, 269I, 269K, 269L, 269M,269N, 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270G, 270H, 270I,270L, 270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y, 271A, 271D, 271E,271F, 271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T,271V, 271W, 271Y, 272D, 272F, 272G, 272H, 272I, 272K, 272L, 272M, 272P,272R, 272S, 272T, 272V, 272W, 272Y, 273I, 274D, 274E, 274F, 274G, 274H,274I, 274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L, 275W,276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P, 276R, 276S, 276T,276V, 276W, 276Y, 278D, 278E, 278G, 278H, 278I, 278K, 278L, 278M, 278N,278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P, 280W,281D, 281E, 281K, 281N, 281P, 281Q, 281Y, 282E, 282G, 282K, 282P, 282Y,283G, 283H, 283K, 283L, 283P, 283R, 283Y, 284D, 284E, 284L, 284N, 284Q,284T, 284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y,288D, 288E, 288Y, 290D, 290H, 290L, 290N, 290W, 291D, 291E, 291G, 291H,291I, 291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L,293M, 293N, 293P, 293R, 293S, 293T, 293V, 293W, 293Y, 294F, 294G, 294H,294I, 294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D,295E, 295F, 295G, 295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V,295W, 295Y, 296A, 296D, 296E, 296G, 296H, 296I, 296K, 296L, 296M, 296N,296Q, 296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G, 297H, 297I, 297K,297L, 297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 298A, 298D,298E, 298F, 298H, 298I, 298K, 298M, 298N, 298Q, 298R, 298T, 298W, 298Y,299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N, 299P,299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H, 300K,300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E, 301H,301Y, 302I, 303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E, 305T,305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F, 320G,320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F,322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y, 323I, 324D, 324F,324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A,325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R,325S, 325T, 325V, 325W, 325Y, 326E, 326I, 326L, 326P, 326T, 327D, 327E,327F, 327H, 327I, 327K, 327L, 327M, 327N, 327P, 327R, 327S, 327T, 327V,327W, 327Y, 328A, 328D, 328E, 328F, 328G, 328H, 328I, 328K, 328M, 328N,328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F, 329G,329H, 329I, 329K, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329W,329Y, 330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R, 330S,330T, 330V, 330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q, 331R,331T, 331V, 331W, 331Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M,332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 333A, 333F, 333H,333I, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 334I, 334L, 334P, 334T,335D, 335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S, 335V,335W, 335Y, 336E, 336K, 336Y, 337E, 337H, and 337N, wherein numbering isaccording to the EU index.

In further aspects, the amino acid modification is 332E. The secondamino acid modification can be selected from the group consisting of:236A, 239D, 332E, 268D, 268E, 330Y, and 330L. In certain embodiments,the second amino acid modification is 239D.

In other aspects, the modification is a glycoform modification thatreduces the level of fucose relative to the parent antibody. In stillother aspects, the invention is directed to a composition including aplurality of glycosylated antibodies, wherein about 80-100% of theglycosylated antibodies in the composition comprise a mature corecarbohydrate structure which lacks fucose.

In a further embodiment, the antibody reduces binding to FcγRIIb ascompared to the parent anti-CD40 antibody.

In another aspect, the invention is directed to an antibody that bindsCD40, includes a heavy chain and/or a light chain, and has an increasedaffinity to the FcγRIIIa receptor as compared to the parent antibody.The heavy chain has a CDR1 comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 16, 22, and 28, a CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 17, 23, and 29, and a CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 18, 24, and 30. Thelight chain has a CDR1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 19, 25, and 31, a CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:20, 26, and 32, and a CDR3 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 21, 27, and 33.

In further variations, the antibody has a variable heavy chain sequenceselected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16-19and/or a variable light chain sequence selected from the groupconsisting of SEQ ID NOs: 11, 13, 15 and 20-22.

In various additional aspects, the invention is directed to a nucleicacid sequence encoding any of the antibodies disclosed herein.

In further aspects, the invention is directed to a method of treating aB-cell related disease by administering an antibody according to claim1. In certain variations, the disease is selected from non-Hodgkin'slymphomas (NHL), chronic lymphocytic leukemia (CLL), B-cell acutelymphoblastic leukemia/lymphoma (B-ALL), mantle cell lymphoma (MCL), andmultiple myeloma (MM). In certain aspects, the disease is an autoimmunedisease, such as rheumatoid arthritis (RA), systemic lupus erythematosus(SLE or lupus), multiple sclerosis, Sjogren's syndrome, and idiopathicthrombocytopenia purpura (ITP).

In further aspects, the invention is directed to a compositioncomprising an antibody described herein and an acceptable carrier.

In one aspect, the invention is directed toward an antibody that bindsCD40, wherein the antibody comprises a means for optimizing effectorfunction. In one embodiment, the means allows for antibody binding withincreased affinity to the FcγRIIIa receptor as compared to the parentantibody. In another embodiment, the means is an amino acidmodification. In some embodiments, the means is a positional means foroptimizing effector function, e.g., modification of an amino acid at oneor more of the following heavy chain constant region positions: 221,222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318,320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, and 337, wherein numbering is according to the EU index. Inother embodiments, the means is a substitutional means for optimizingeffector function, e.g., one or more of the following amino acidsubstitutions in the heavy chain constant region positions: 221K, 221Y,222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K,227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E, 231G, 231K,231P, 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I,233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A,234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P, 234Q, 234R,234S, 234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F, 235G, 235H, 235I,235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W, 235Y, 236A,236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P, 236Q, 236R,236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I, 237K, 237L,237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y, 238D, 238E,238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R, 238S, 238T,238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K, 239L, 239M,239N, 239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240A, 240I, 240M, 240T,241D, 241E, 241L, 241R, 241S, 241W, 241Y, 243E, 243H, 243L, 243Q, 243R,243W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V, 249H, 249Q,249Y, 255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 260H, 260Y, 262A, 262E,262F, 262I, 262T, 263A, 263I, 263M, 263T, 264A, 264D, 264E, 264F, 264G,264H, 264I, 264K, 264L, 264M, 264N, 264P, 264Q, 264R, 264S, 264T, 264W,264Y, 265F, 265G, 265H, 265I, 265K, 265L, 265M, 265N, 265P, 265Q, 265R,265S, 265T, 265V, 265W, 265Y, 266A, 266I, 266M, 266T, 267D, 267E, 267F,267H, 267I, 267K, 267L, 267M, 267N, 267P, 267Q, 267R, 267T, 267V, 267W,267Y, 268D, 268E, 268F, 268G, 268I, 268K, 268L, 268M, 268P, 268Q, 268R,268T, 268V, 268W, 269F, 269G, 269H, 269I, 269K, 269L, 269M, 269N, 269P,269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270G, 270H, 270I, 270L, 270M,270P, 270Q, 270R, 270S, 270T, 270W, 270Y, 271A, 271D, 271E, 271F, 271G,271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V, 271W,271Y, 272D, 272F, 272G, 272H, 272I, 272K, 272L, 272M, 272P, 272R, 272S,272T, 272V, 272W, 272Y, 273I, 274D, 274E, 274F, 274G, 274H, 274I, 274L,274M, 274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L, 275W, 276D, 276E,276F, 276G, 276H, 276I, 276L, 276M, 276P, 276R, 276S, 276T, 276V, 276W,276Y, 278D, 278E, 278G, 278H, 278I, 278K, 278L, 278M, 278N, 278P, 278Q,278R, 278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P, 280W, 281D, 281E,281K, 281N, 281P, 281Q, 281Y, 282E, 282G, 282K, 282P, 282Y, 283G, 283H,283K, 283L, 283P, 283R, 283Y, 284D, 284E, 284L, 284N, 284Q, 284T, 284Y,285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y, 288D, 288E,288Y, 290D, 290H, 290L, 290N, 290W, 291D, 291E, 291G, 291H, 291I, 291Q,291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L, 293M, 293N,293P, 293R, 293S, 293T, 293V, 293W, 293Y, 294F, 294G, 294H, 294I, 294K,294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D, 295E, 295F,295G, 295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V, 295W, 295Y,296A, 296D, 296E, 296G, 296H, 296I, 296K, 296L, 296M, 296N, 296Q, 296R,296S, 296T, 296V, 297D, 297E, 297F, 297G, 297H, 297I, 297K, 297L, 297M,297P, 297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 298A, 298D, 298E, 298F,298H, 298I, 298K, 298M, 298N, 298Q, 298R, 298T, 298W, 298Y, 299A, 299D,299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N, 299P, 299Q, 299R,299S, 299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H, 300K, 300M, 300N,300P, 300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E, 301H, 301Y, 302I,303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E, 305T, 305Y, 313F,317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F, 320G, 320H, 320I,320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F, 322G, 322H,322I, 322P, 322S, 322T, 322V, 322W, 322Y, 323I, 324D, 324F, 324G, 324H,324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A, 325D, 325E,325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R, 325S, 325T,325V, 325W, 325Y, 326E, 326I, 326L, 326P, 326T, 327D, 327E, 327F, 327H,327I, 327K, 327L, 327M, 327N, 327P, 327R, 327S, 327T, 327V, 327W, 327Y,328A, 328D, 328E, 328F, 328G, 328H, 328I, 328K, 328M, 328N, 328P, 328Q,328R, 328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F, 329G, 329H, 329I,329K, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329W, 329Y, 330E,330F, 330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R, 330S, 330T, 330V,330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q, 331R, 331T, 331V,331W, 331Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M, 332N, 332P,332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 333A, 333F, 333H, 333I, 333L,333M, 333P, 333T, 333Y, 334A, 334F, 334I, 334L, 334P, 334T, 335D, 335F,335G, 335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S, 335V, 335W, 335Y,336E, 336K, 336Y, 337E, 337H, and 337N, wherein numbering is accordingto the EU index.

In other embodiments, the means for optimizing effector function is apositional means, e.g., modification of an amino acid at one or more ofthe following positions: 221, 222, 223, 224, 225, 228, 230, 231, 232,240, 244, 245, 247, 262, 263, 266, 271, 273, 275, 281, 284, 291, 299,302, 304, 313, 323, 325, 328, 332, 336, wherein the positional numberingis according to the EU index. In some embodiments, the means foroptimizing effector function is a substitutional means, e.g., one ormore of the following substitutions: 221K, 221Y, 222E, 222Y, 223E, 223K,224E, 224Y, 225E, 225K, 225W, 228E, 228G, 228K, 228Y, 230A, 230E, 230G,230Y, 231E, 231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 240A, 240I,240M, 240T, 244H, 245A, 247G, 247V, 262A, 262E, 262F, 262I, 262T, 263A,263I, 263M, 263T, 266A, 266I, 266M, 266T, 271A, 271D, 271E, 271F, 271G,271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V, 271W,271Y, 273I, 275L, 275W, 281D, 281E, 281K, 281N, 281P, 281Q, 281Y, 284D,284E, 284L, 284N, 284Q, 284T, 284Y, 291D, 291E, 291G, 291H, 291I, 291Q,291T, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N,299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 304D, 304H, 304L, 304N, 304T,313F, 323I, 325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M,325P, 325Q, 325R, 325S, 325T, 325V, 325W, 325Y, 328A, 328D, 328E, 328F,328G, 328H, 328I, 328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T, 328V,328W, 328Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M, 332N, 332P,332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 336E, 336K, and 336Y. In otherembodiments, the means for optimizing effector function includes amodification at a second amino acid, e.g., at a position selected fromthe group consisting of 221, 222, 223, 224, 225, 227, 228, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246,247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286,288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, and 337, wherein numbering isaccording to the EU index. For example, the means for optimizingeffector function may include a substitution at a second amino acid,e.g., one or more of the following substitutions: 221K, 221 Y, 222E,222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y,228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E, 231G, 231K, 231P,231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K,233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 234D,234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P, 234Q, 234R, 234S,234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F, 235G, 235H, 235I, 235K,235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W, 235Y, 236A, 236D,236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P, 236Q, 236R, 236S,236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I, 237K, 237L, 237M,237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y, 238D, 238E, 238F,238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R, 238S, 238T, 238V,238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K, 239L, 239M, 239N,239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240A, 240I, 240M, 240T, 241D,241E, 241L, 241R, 241S, 241W, 241Y, 243E, 243H, 243L, 243Q, 243R, 243W,243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V, 249H, 249Q, 249Y,255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 260H, 260Y, 262A, 262E, 262F,262I, 262T, 263A, 263I, 263M, 263T, 264A, 264D, 264E, 264F, 264G, 264H,264I, 264K, 264L, 264M, 264N, 264P, 264Q, 264R, 264S, 264T, 264W, 264Y,265F, 265G, 265H, 265I, 265K, 265L, 265M, 265N, 265P, 265Q, 265R, 265S,265T, 265V, 265W, 265Y, 266A, 266I, 266M, 266T, 267D, 267E, 267F, 267H,267I, 267K, 267L, 267M, 267N, 267P, 267Q, 267R, 267T, 267V, 267W, 267Y,268D, 268E, 268F, 268G, 268I, 268K, 268L, 268M, 268P, 268Q, 268R, 268T,268V, 268W, 269F, 269G, 269H, 269I, 269K, 269L, 269M, 269N, 269P, 269R,269S, 269T, 269V, 269W, 269Y, 270F, 270G, 270H, 270I, 270L, 270M, 270P,270Q, 270R, 270S, 270T, 270W, 270Y, 271A, 271D, 271E, 271F, 271G, 271H,271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y,272D, 272F, 272G, 272H, 272I, 272K, 272L, 272M, 272P, 272R, 272S, 272T,272V, 272W, 272Y, 273I, 274D, 274E, 274F, 274G, 274H, 274I, 274L, 274M,274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L, 275W, 276D, 276E, 276F,276G, 276H, 276I, 276L, 276M, 276P, 276R, 276S, 276T, 276V, 276W, 276Y,278D, 278E, 278G, 278H, 278I, 278K, 278L, 278M, 278N, 278P, 278Q, 278R,278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P, 280W, 281D, 281E, 281K,281N, 281P, 281Q, 281Y, 282E, 282G, 282K, 282P, 282Y, 283G, 283H, 283K,283L, 283P, 283R, 283Y, 284D, 284E, 284L, 284N, 284Q, 284T, 284Y, 285D,285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y, 288D, 288E, 288Y,290D, 290H, 290L, 290N, 290W, 291D, 291E, 291G, 291H, 291I, 291Q, 291T,292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L, 293M, 293N, 293P,293R, 293S, 293T, 293V, 293W, 293Y, 294F, 294G, 294H, 294I, 294K, 294L,294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D, 295E, 295F, 295G,295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V, 295W, 295Y, 296A,296D, 296E, 296G, 296H, 296I, 296K, 296L, 296M, 296N, 296Q, 296R, 296S,296T, 296V, 297D, 297E, 297F, 297G, 297H, 297I, 297K, 297L, 297M, 297P,297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 298A, 298D, 298E, 298F, 298H,298I, 298K, 298M, 298N, 298Q, 298R, 298T, 298W, 298Y, 299A, 299D, 299E,299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N, 299P, 299Q, 299R, 299S,299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H, 300K, 300M, 300N, 300P,300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E, 301H, 301Y, 302I, 303D,303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E, 305T, 305Y, 313F, 317E,317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F, 320G, 320H, 320I, 320L,320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F, 322G, 322H, 322I,322P, 322S, 322T, 322V, 322W, 322Y, 323I, 324D, 324F, 324G, 324H, 324I,324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A, 325D, 325E, 325F,325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R, 325S, 325T, 325V,325W, 325Y, 326E, 326I, 326L, 326P, 326T, 327D, 327E, 327F, 327H, 327I,327K, 327L, 327M, 327N, 327P, 327R, 327S, 327T, 327V, 327W, 327Y, 328A,328D, 328E, 328F, 328G, 328H, 328I, 328K, 328M, 328N, 328P, 328Q, 328R,328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F, 329G, 329H, 329I, 329K,329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329W, 329Y, 330E, 330F,330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R, 330S, 330T, 330V, 330W,330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q, 331R, 331T, 331V, 331W,331Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M, 332N, 332P, 332Q,332R, 332S, 332T, 332V, 332W, 332Y, 333A, 333F, 333H, 333I, 333L, 333M,333P, 333T, 333Y, 334A, 334F, 334I, 334L, 334P, 334T, 335D, 335F, 335G,335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S, 335V, 335W, 335Y, 336E,336K, 336Y, 337E, 337H, and 337N, wherein numbering is according to theEU index.

In another embodiment, the means for optimizing effector function is theamino acid modification 332E. In some embodiments, the means foroptimizing effector function is the amino acid modification 332E and oneor more of the following amino acid modifications: 236A, 239D, 332E,268D, 268E, 330Y, and 330L, e.g., 239D.

In other embodiments, an antibody of the invention has a reduced levelof fucose relative to the parent antibody. For example, a composition ofthe invention may comprise a plurality of glycosylated antibodies,wherein about 80-100% of the glycosylated antibodies have a reducedlevel of fucose.

In other embodiments, an antibody of the invention comprises a means toreduce binding to FcγRIIb as compared to the parent anti-CD40 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings further illustrate aspects of the invention, andare not meant to constrain the present invention to any particularapplication or theory of operation.

FIG. 1. Sequences of the natural antibody constant regions, includingthe kappa constant light chain, and the gamma constant heavy chains forIgG1, IgG2, IgG3, and IgG4. Also provided is the sequence of a HybridIgG constant chain, and a Hybrid IgG constant chain comprising thesubstitutions 239D and 1332E.

FIG. 1 b. Alignment of the amino acid sequences of the human IgGimmunoglobulins IgG1, IgG2, IgG3, and IgG4 (SEQ ID NO:2-5,respectively). FIG. 1 b provides the sequences of the CH1 (Cγ1) andhinge domains, and FIG. 1 c provides the sequences of the CH2 (Cγ2) andCH3 (Cγ3) domains. Positions are numbered according to the EU index ofthe IgG1 sequence, and differences between IgG1 and the otherimmunoglobulins IgG2, IgG3, and IgG4 are shown in gray. Allotypicpolymorphisms exist at a number of positions, and thus slightdifferences between the presented sequences and sequences in the priorart may exist. The possible beginnings of the Fc region are labeled,defined herein as either EU position 226 or 230.

FIG. 1 c. Alignment of the amino acid sequences of the human IgGimmunoglobulins IgG1, IgG2, IgG3, and IgG4. FIG. 1 c provides thesequences of the CH1 (Cγ1), hinge domain, CH2 (Cγ2) domain and CH3 (Cγ3)domain. Positions are numbered according to the EU index of the IgG1sequence, and differences between IgG1 and the other immunoglobulinsIgG2, IgG3, and IgG4 are shown in grey. Polymorphisms exist at a numberof positions (Kim et al., 2001, J. Mol. Evol. 54:1-9, incorporatedherein by reference in its entirety), and thus slight differencesbetween the presented sequences and sequences in the prior art mayexist. The possible beginnings of the Fc region are labeled, definedherein as either EU position 226 or 230.

FIGS. 2 a and 2 b. The common haplotypes of the gamma chain of humanIgG1 (FIG. 2 a) and IgG2 (FIG. 2 b) showing the positions and therelevant amino acid substitutions.

FIG. 3. Embodiments of receptor binding profiles that include increasesto, reductions to, or no effect on the binding to various receptors,where such changes may be beneficial in certain contexts.

FIG. 4: Amino acid sequences of the CD40 antigen targeted by theantibodies of the invention. FIG. 4 provides the sequence of bothisoforms of Homo sapiens CD40.

FIGS. 5 a-5 d. Sequences encoding the murine anti-CD40 antibody variableregions. FIG. 5 a provides the light and heavy chain variable regionsequences of the anti-CD40 antibodies S2C6, G28-5, and 5D12,respectively. CDRs are underlined. FIGS. 5 b-5 d describe the CDRsequences of S2C6, G28-5, and 5D12, respectively.

FIGS. 6 a-6 c. ADCC assay comparing WT IgG1, Hybrid S239D/I332E(effector function enhanced) anti-CD40 antibodies, and the anti-CD20antibody rituximab on the Burkitt's Lymphoma cell lines Daudi and Raji,and the multiple myeloma cell line RPMI8226. FIG. 6 a shows results forS2C6, FIG. 6 b shows results for 5D12, and FIG. 6 c shows results forG28-5.

FIG. 7. Platelet activation assay with anti-CD40 antibody S2C6. Samplesincluded in the assay were S2C6 IgG1, S2C6 Hybrid S239D/I332E, positivecontrol antibody IV.3 (mouse anti-human IgG FcgRIIa specific antibody),and isotype control antibody.

FIG. 8. Anti-proliferation assay of anti-CD40 antibodies on theBurkitt's Lymphoma cell line HS-Sultan. Samples included in the assaywere 5D12 IgG1, S2C6 IgG1, and human IgG with and without CD40 ligandand cells only were used as controls.

FIG. 9. Amino acid sequences of humanized heavy chain S2C6 variants.

FIG. 10. Amino acid sequences of humanized light chain S2C6 variants.

FIG. 11. ADCC of humanized S2C6 templates incubated at 60° C. at 0 and48 hrs on RAMOS cells in order to assess the relative stability of eachtemplate.

FIGS. 12 a-12 d. ADCC of several humanized S2C6 templates on threedifferent cell lines. FIG. 12 a shows ADCC with S2C6 H3L1 HybridS239D/I332E, rituximab (anti-CD20), and isotype control (HybridS239D/I332E) on the Burkitt's Lymphoma cell line Ramos. FIG. 12 b showsADCC with S2C6 H4L3 Hybrid S239D/I332E, rituximab (anti-CD20), andisotype control (Hybrid S239D/I332E) on the Burkitt's Lymphoma cell lineNamalwa. FIG. 12 c shows ADCC with S2C6 H1L1 Hybrid S239D/I332E, S2C6H1L1 IgG1, and isotype control (Hybrid S239D/I332E) on theB-lymphoblastoid cell line IM-9. FIG. 12 d shows ADCC with S2C6 H1L1Hybrid S239D/I332E, S2C6 H1L1 IgG1, and isotype control (HybridS239D/I332E) on the multiple myeloma cell line RPMI8226.

FIG. 13. Survival of SCID mice in a model of Ramos Burkitt's lymphomaafter treatment with several humanized S2C6 templates. FIG. 13 shows aKaplan-Myer graph delineating the survival of SCID mice injectedintravenously (i.v.) with 5×10⁶ human Ramos Burkitt's lymphoma cells andtreated with PBS, isotype control (Hybrid S239D/I332E Fc), 6 mg/kg S2C6H1L1 Hybrid S239D/I332E (anti-CD40), 2 mg/kg S2C6 H1L1 HybridS239D/I332E (anti-CD40), or 0.6 mg/kg S2C6 H1L1 Hybrid S239D/I332E(anti-CD40).

DETAILED DESCRIPTION OF THE INVENTION

B-Cell Disorders:

Because of their critical role in regulating the immune system,disregulation of B cells is associated with a variety of disorders.B-cell disorders, also referred to herein as B-cell related diseases,are divided into excessive or uncontrolled proliferation (lymphomas,leukemias), and defects of B-cell development/immunoglobulin production(immunodeficiencies). The majority (80%) of lymphoma cases are of B-cellorigin. These include non-Hodgkin's lymphoma (NHL), acute lymphoblasticleukemia (ALL), and autoimmune related diseases.

NHL is a heterogeneous malignancy originating from lymphocytes. In theUnited States (U.S.), the incidence is estimated at 65,000/year withmortality of approximately 20,000 (American Cancer Society, 2006; andSEER Cancer Statistics Review). The disease can occur in all ages, theusual onset begins in adults over 40 years of age, with the incidenceincreasing with age. NHL is characterized by a clonal proliferation oflymphocytes that accumulate in the lymph nodes, blood, bone marrow andspleen, although any major organ may be involved.

The diagnosis and histologic characterization of NHL is made using acombination of morphologic and immunophenotype criteria. The currentclassification system used by pathologists and clinicians is the WorldHealth Organization (WHO) Classification of Tumours, which organizes NHLinto precursor and mature B-cell or T-cell neoplasms. The PDQ iscurrently dividing NHL as indolent or aggressive for entry into clinicaltrials. For consistency the present document will also use a similardivision. The indolent NHL group is comprised primarily of follicularsubtypes, small lymphocytic lymphoma, MALT, and marginal zone; indolentencompasses approximately 50% of newly diagnosed B-cell NHL patients.Aggressive NHL includes patients with histologic diagnoses of primarilydiffuse large B cell (40% of all newly diagnosed patients have diffuselarge cell), Burkitt's, and mantle cell. The clinical course of NHL ishighly variable. A major determinant of clinical course is thehistologic subtype. Most indolent types of NHL are considered to beincurable diseases. Patients respond initially to either chemotherapy orantibody therapy and most will relapse. Studies to date have notdemonstrated an improvement in survival with early intervention. Inasymptomatic patients, it is acceptable to “watch and wait” until thepatient becomes symptomatic or the disease pace appears to beaccelerating. Over time, the disease may transform to a more aggressivehistology. The median survival is 8 to 10 years, and indolent patientsoften receive 3 or more treatments during the treatment phase of theirdisease. Initial treatment of the symptomatic indolent NHL patienthistorically has been combination chemotherapy. The most commonly usedagents include: cyclophosphamide, vincristine and prednisone (CVP);cyclophosphamide, adriamycin, vincristine, prednisone (CHOP); or thepurine analog, fludarabine. Approximately 70% to 80% of patients willrespond to their initial chemotherapy, duration of remissions last onthe order of 2-3 years. Ultimately the majority of patients relapse. Thediscovery and clinical use of the anti-CD20 antibody, rituximab, hasprovided significant improvements in response and survival rate. Thecurrent standard of care for most patients is rituximab+CHOP (R-CHOP) orrituximab+CVP (R-CVP). Interferon is approved for initial treatment ofNHL in combination with alkylating agents, but has limited use in theU.S.

Rituximab therapy has been shown to be efficacious in several types ofNHL, and is currently approved as a first line treatment for bothindolent (follicular lymphoma) and aggressive NHL (diffuse large B celllymphoma). However, there are significant limitations of anti-CD20monoclonal antibody (mAb), including primary resistance (50% response inrelapsed indolent patients), acquired resistance (50% response rate uponre-treatment), rare complete response (2% complete response rate inrelapsed population), and a continued pattern of relapse. Finally, manyB cells do not express CD20, and thus many B-cell disorders are nottreatable using anti-CD20 antibody therapy. Antibodies against antigensother than CD20 may have anti-lymphoma effects that could overcomeanti-CD20 resistance or augment the activity of anti-CD20 therapy.

In addition to NHL there are several types of leukemias that result fromdisregulation of B cells. Chronic lymphocytic leukemia (also known as“chronic lymphoid leukemia” or “CLL”), is a type of adult leukemiacaused by an abnormal accumulation of B lymphocytes. In CLL, themalignant lymphocytes may look normal and mature, but they are not ableto cope effectively with infection. CLL is the most common form ofleukemia in adults. Men are twice as likely to develop CLL as women.However, the key risk factor is age. Over 75% of new cases are diagnosedin patients over age 50. More than 10,000 cases are diagnosed every yearand the mortality is almost 5,000 a year (American Cancer Society, 2006;and SEER Cancer Statistics Review).

CLL is an incurable disease but progresses slowly in most cases. Manypeople with CLL lead normal and active lives for many years. Because ofits slow onset, early-stage CLL is generally not treated since it isbelieved that early CLL intervention does not improve survival time orquality of life. Instead, the condition is monitored over time. InitialCLL treatments vary depending on the exact diagnosis and the progressionof the disease. There are dozens of agents used for CLL therapy.Although the purine analogue fludarabine was shown to give superiorresponse rates than chlorambucil as primary therapy, there is noevidence that early use of fludarabine improves overall survival.Combination chemotherapy regimens such as fludarabine withcyclophosphamide, FCR (fludarabine, cyclophosphamide and rituximab) andCHOP are effective in both newly-diagnosed and relapsed CLL. Allogeneicbone marrow (stem cell) transplantation is rarely used as a first-linetreatment for CLL due to its risk.

“Refractory” CLL is a disease that no longer responds favorably totreatment. In this case more aggressive therapies, including bone marrow(stem cell) transplantation, are considered. The monoclonal antibodyalemtuzumab, directed against CD52, may be used in patients withrefractory, bone marrow-based disease.

Another type of leukemia is acute lymphoblastic leukemia (ALL), alsoknown as acute lymphocytic leukemia. ALL is characterized by theoverproduction and continuous multiplication of malignant and immaturewhite blood cells (also known as lymphoblasts) in the bone marrow.‘Acute’ refers to the undifferentiated, immature state of thecirculating lymphocytes (“blasts”), and that the disease progressesrapidly with life expectancy of weeks to months if left untreated. ALLis most common in childhood with a peak incidence of 4-5 years of age.Children of age 12-16 die more easily from it than those of other ages.Currently, at least 80% of childhood ALL are considered curable. Under4,000 cases are diagnosed every year and the mortality rate is almost1,500 a year (American Cancer Society, 2006; and SEER Cancer StatisticsReview).

Multiple myeloma (MM) is a B-cell malignancy with terminallydifferentiated plasma cells. The disease subsequently attacks bone andbone marrow, which results in multiple tumors and lesions throughout theskeletal system. It is the second most common hematologic malignancy inthe United States, afflicting approximately 55,000 people there alone.The annual incidence rate for multiple myeloma is 3 to 4 cases per100,000 people, making it the most common bone tumor cancer in adults.Current treatment protocols which include a combination ofchemotherapeutic agents yield a complete remission rate of only about5%, with a median survival of approximately 36-48 months from the timeof diagnosis. Treatment regimens are limited by a low cell proliferationrate and the development of multi-drug resistance. Therefore,alternative treatment regimens using therapeutic antibodies targetingsurface antigens on plasma cells would be of great benefit.

Autoimmunity results from a breakdown of self-tolerance involvinghumoral and/or cell-mediated immune mechanisms. The consequences offailure in central and/or peripheral tolerance include survival andactivation of self-reactive B cells and T cells. Examples of autoimmunediseases include, for example, rheumatoid arthritis (RA), systemic lupuserythematosus (SLE or lupus), multiple sclerosis (MS), Sjogren'ssyndrome, and idiopathic thrombocytopenia purpura (ITP). Thepathogenesis of most autoimmune diseases is coupled to the production ofautoantibodies against self antigens, leading to a variety of associatedpathologies. Autoantibodies are produced by terminally differentiatedplasma cells that are derived from naïve or memory B cells. Furthermore,B cells can have other effects on autoimmune pathology, asantigen-presenting cells (APCs) that can interact with and stimulatehelper T cells, further stimulating the cycle of anti-self immuneresponse. Depletion of B cells can have direct impact on the productionof autoantibodies. Indeed, treatment of RA and SLE with B-cell depletiontherapies such as rituximab has been demonstrated to have clinicalbenefit for both disease classes (Edwards & Cambridge, Nat. Rev.Immunol. 2006; Dass et al., Future Rheumatol. 2006; Martin & Chan, Annu.Rev. Immunol. 2006, each incorporated herein it its entirety byreference).

Antibodies as Therapeutics to Treat B-Cell Disorders and Solid Tumors

Monoclonal antibodies are a class of therapeutic proteins that may beused to treat B-cell disorders. A number of favorable properties ofantibodies, including but not limited to specificity for target, abilityto mediate immune effector mechanisms, and long half-life in serum, makeantibodies powerful therapeutics. The present invention describesantibodies against the B-cell and solid tumor antigen CD40.

CD40:

The CD40 antigen is a cell surface glycoprotein which belongs to thetumor necrosis factor receptor (TNF-R) family and is expressed on thesurface of all mature B cells, most mature B-cell malignancies, someearly B-cell acute lymphocytic leukemias, and ˜70% of all solid tumors.CD40 is also expressed on dendritic cells, monocytes, endothelial cells,epithelial cells, fibroblasts, smooth muscle cells, and many human solidtumors, including melanoma and carcinomas. Signaling via CD40 activatesantigen-presenting cells, including B-cells and dendritic cells. CD154,which is the natural ligand of CD40, is expressed on the surface ofactivated T-lymphocytes and provides a large component of T-celltriggering for immune responses. Consequently, CD40 agonists triggerimmune responses against many tumor-associated antigens. Because ofthese properties, and its expression on many B-cell malignancies andsolid tumors, therapeutic anti-CD40 antibodies would be of great valuein treating disease.

CD40 is a 50-kd type I transmembrane protein of the TNFR family thatincludes TNFR-1, TNFR-2, CD30, CD27, 4-1BB, OX40, and Fas. Thegene-encoding CD40 is located at chromosome 20 in humans and chromosome2 in mice. Correlated with its immune regulatory function in variouscell types, CD40 is expressed widely on B cells, DCs, monocytes andmacrophages, thymic epithelial cells, endothelial cells, mast cells,fibroblasts, and smooth muscle cells. CD40 ligand (CD154) is produced asa type II transmembrane protein with an apparent molecular mass of 32 to33 kd. It is a member of the TNF family, which also includes TNF-α,CD153, CD70, 4-1BBL, OX40L, and FasL. Soluble forms of CD154 exist as 31or 18-kd proteins, both of which retain the biologic activities of themembrane-bound CD154, and can potentially act as cytokines on distalCD40+ cells. The gene-encoding CD154 is located on the X chromosome inboth humans and mice. Mutations in the CD154 gene result in the X-linkedhyper-immunoglobulin (Ig) M syndrome in humans. CD154 expression,originally thought to be restricted to activated T cells, mainly of theCD4 lineage, has now been identified on CD8+T cells, B cells,eosinophils, mast cells, basophils, DCs, and other cell types. Recently,platelets were found to express CD154 as well.

Originally identified as the molecular interaction between T and Bcells, the CD154-CD40 pathway plays a key role in regulatingthymus-dependent humoral responses. Indeed, patients with X-linkedhyper-IgM syndrome have elevated IgM levels but low levels of IgA, IgG,and IgE; are devoid of the germinal centers; and are unable to mountthymus-dependent humoral responses. In mice, CD154-CD40 signaling wasshown to be critical in regulating B-cell proliferation, Ig production,Ig class switching, rescue of B cells from apoptosis, germinal centerformation, generation of B-cell memory, and clonal expansion anddeletion of B cells.

Most APC at the resting stage express low levels of costimulatorymolecules, such as CD80 or CD86. CD40 ligation by CD154 from T cellsrepresents a key step in APC activation to enhance costimulatorymolecule expression, which enables them to be competent to fullyactivate T cells. Induction of proinflammatory cytokines, includinginterleukin (IL) 12 by DCs, is another outcome of this APC interaction,which is critical for the development of TH1-type immune responses. Thusblockade of the CD154-CD40 pathway will result in a deficiency of APCactivation, which will obviously also lead to the global failure ofT-cell activation. However, this indirect impact on T cells is not theonly reason for ineffective T-cell responses after CD154 blockade. It isnow realized that CD154-CD40 interaction is bidirectional. Because invitro CD154 ligation on T cells significantly enhances cytokineproduction, particularly of the TH2 type, cross-linking of CD154 on CD4T cells in vivo contributes to the generation of helper function andgerminal center. CD154-CD40 interaction provides the molecular basis forCD4 help in activation of cytotoxic CD8+ T cells, with DCs serving as abridge between the 2 cell types. Although the molecular details of this3-cell interaction have not been fully elucidated, it is now clear thatactivated CD4+ T cells stimulate DCs via CD154-CD40 and enable DCs tosubsequently activate CD8+ T cells to differentiate into cytotoxiceffectors.

CD40 is expressed on many nonhematopoietic cells, particularly under theproinflammatory conditions. Cross-linking of CD40 on vascularendothelial cells activates these cells and increases expression ofadhesion molecules, such as CD62E, CD106, CD54, and chemokines andcytokines, including IL-6 and IL-8. This may promote extravasation andaccumulation of activated T cells at the sites of inflammation. Inaddition to activated T cells, platelets may provide another source ofendothelial activation. Indeed, thrombin-activated platelets rapidlyup-regulate CD154 expression, which may then interact with CD40 onendothelial cells and thus contribute to chemotactic effects fromendothelial activation or damage, independently of T cells. This mayhave contributed, at least in part, to unexpected thromboemboliccomplications recently detected in primates and humans treated with someCD154 antibodies.

CD154/CD40 signaling is also crucially important for controllingimmunity and inflammation. The emerging picture indicates that ligationof the receptor CD40 via CD154, most potently in its trimeric form,functions in two ways. CD154 modulates physiologic processes, such as Tcell-mediated effector functions and general immune responses requiredfor appropriate host defense, but also triggers the expression ofpro-inflammatory mediators, such as cytokines, adhesion molecules, andmatrix degrading activities, all of which are associated with thepathogenesis of chronic inflammatory diseases, e.g., autoimmunedisorders, arthritis, atherosclerosis, and cancer. Accordingly,CD40/CD154 interactions have advanced as a potential therapeutic targetfor these diseases, whereby two opposing strategies, interruption aswell as enhancement of CD40 signaling, are explored for beneficialoutcomes. Besides their crucial role in T-cell-dependent humoralimmunity, CD40-CD40L interactions have thus been implicated inautoimmune diseases, such as atherosclerosis, asthma, systemic lupuserythematosus, multiple sclerosis, graft versus host disease,experimental autoimmune encephalitis and rheumatoid arthritis.

Fc Optimization of Antibodies May Provide Improved Clinical Performance

The clinical success of antibodies directed against CD40 will dependheavily on their potential mechanism(s) of action. There are a number ofpossible mechanisms by which antibodies mediate cellular effects,including anti-proliferation via blockage of needed growth pathways,intracellular signaling leading to apoptosis, enhanced down regulationand/or turnover of receptors, complement-dependent cytotoxicity (CDC),antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependentcell-mediated phagocytosis (ADCP) and promotion of an adaptive immuneresponse (Cragg at al., 1999, Curr Opin Immunol 11:541-547; Glennie etal., 2000, Immunol Today 21:403-410, each incorporated herein it itsentirety by reference). Antibody efficacy may be due to a combination ofthese mechanisms, and their relative importance in clinical therapy foroncology appears to be cancer dependent.

The importance of FcγR-mediated effector functions for the activity ofsome antibodies has been demonstrated in mice (Clynes et al., 1998, ProcNatl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med 6:443-446,each incorporated herein it its entirety by reference), and fromobserved correlations between clinical efficacy in humans and theirallotype of high (V158) or low (F158) affinity polymorphic forms ofFcγRIIIa (Cartron et al., 2002, Blood 99:754-758; Weng & Levy, 2003,Journal of Clinical Oncology, 21:3940-3947, each incorporated herein itits entirety by reference). Together these data suggest that an antibodythat is optimized for binding to certain FcγRs may better mediateeffector functions, and thereby destroy target cells more effectively inpatients. Thus a promising means for enhancing the anti-tumor potency ofantibodies is via enhancement of their ability to mediate cytotoxiceffector functions such as ADCC, ADCP, and CDC. Additionally, antibodiescan mediate anti-tumor mechanism via growth inhibitory or apoptoticsignaling that may occur when an antibody binds to its target on tumorcells. Such signaling may be potentiated when antibodies are presentedto tumor cells bound to immune cells via FcγR. Therefore increasedaffinity of antibodies to FcγRs may result in enhancedanti-proliferative effects.

Some success has been achieved at modifying antibodies with selectivelyenhanced binding to FcγRs to provide enhanced effector function.Antibody engineering for optimized effector function has been achievedusing amino acid modifications (see for example U.S. Ser. No. 10/672,280and U.S. Ser. No. 11/124,620 and references cited therein, eachincorporated herein it its entirety by reference), and engineeredglycoforms (see for example Umana et al., 1999, Nat Biotechnol17:176-180; Shinkawa et al., 2003, J Biol Chem 278:3466-3473,Yamane-Ohnuki et al., 2004, Biotechnology and Bioengineering87(5):614-621, each incorporated herein it its entirety by reference).

Unfortunately, it is not known a priori which mechanisms of action maybe optimal for a given target antigen. Furthermore, it is not knownwhich antibodies may be capable of mediating a given mechanism of actionagainst a target cell. In some cases a lack of antibody activity, eitherFv-mediated or Fc-mediated, may be due to the targeting of an epitope onthe target antigen that is poor for mediating such activity. In othercases, the targeted epitope may be amenable to a desired Fv-mediated orFc-mediated activity, yet the affinity (affinity of the Fv region forantigen or affinity of the Fc region for Fc receptors) may beinsufficient. Towards addressing this problem, the present inventiondescribes modifications to anti-CD40 antibodies that provide optimizedFv- and Fc-mediated activities.

In order that the invention may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position. Thus “amino acid” asused herein is both naturally occurring and synthetic amino acids. Forexample, homophenylalanine, citrulline and noreleucine are consideredamino acids for the purposes of the invention. “Amino acid” alsoincludes imino acid residues such as proline and hydroxyproline. Theside chain may be in either the (R) or the (S) configuration. In aembodiment, the amino acids are in the (S) or L-configuration. Ifnon-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradation.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (υ), delta (δ), gamma (γ), sigma (σ), and alpha(α) which encode the IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE,and IgA (IgA1 and IgA2) isotypes respectively. Antibody herein is meantto include full length antibodies and antibody fragments, and may referto a natural antibody from any organism, an engineered antibody, or anantibody generated recombinantly for experimental, therapeutic, or otherpurposes.

By “B cell” or “B lymphocyte” as used herein is meant a type oflymphocyte developed in bone marrow that circulates in the blood andlymph, and provides humoral immunity. B cells recognize free antigenmolecules and differentiate or mature into plasma cells that secreteimmunoglobulin (antibodies) that inactivate the antigens. Memory cellsare also generated that make the specific Immunoglobulin (antibody) onsubsequent encounters with such antigen. B cells are also known as “Betacells” in the islet of Langerhans.

By “B-cell antigen” or “B-cell marker” as used herein is meant anyprotein that is expressed on B cells. B-cell markers of the inventioninclude CD40.

By “CD40” as used herein is meant the protein encoded by the genedesignated CD40. CD40 is also known as Tumor necrosis factor receptorsuperfamily member 5 (TNFRSF5), CD40L receptor, CD154 receptor, B-cellsurface antigen CD40, CDw40, and Bp50. Human CD40 is designatedGeneID:958 by Entrez Gene (Maglott et al., 2005, Nucleic Acids Res33(Database Issue):D54-D58, and HGNC:11919 by HUGO (The Human GenomeOrganisation) Gene Nomenclature Committee (HGNC) (Wain et al., 2004,Genew: the Human Gene Nomenclature Database, 2004 updates, Nucleic AcidsRes 32 Database issue:D255-7. The use of CD40 herein is meant toencompass all known or as yet undiscovered alleles and polymorphic formsof CD40. The sequence of human CD40 antigen used in the present study isprovided in FIG. 4, SEQ ID NO: 8.

By “CDC” or “complement dependent cytotoxicity” as used herein is meantthe reaction wherein one or more complement protein components recognizebound antibody on a target cell and subsequently cause lysis of thetarget cell.

By “constant region” of an antibody as defined herein is meant theregion of the antibody that is encoded by one of the light or heavychain immunoglobulin constant region genes. By “constant light chain” or“light chain constant region” as used herein is meant the region of anantibody encoded by the kappa (Cκ) or lambda (Cλ) light chains. Theconstant light chain typically comprises a single domain, and as definedherein refers to positions 108-214 of Cκ or Cλ, wherein numbering isaccording to the EU index. By “constant heavy chain” or “heavy chainconstant region” as used herein is meant the region of an antibodyencoded by the mu, delta, gamma, alpha, or epsilon genes to define theantibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For fulllength IgG antibodies, the constant heavy chain, as defined herein,refers to the N-terminus of the CH1 domain to the C-terminus of the CH3domain, thus comprising positions 118-447, wherein numbering isaccording to the EU index.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include FcγR-mediated effectorfunctions such as ADCC and ADCP, and complement-mediated effectorfunctions such as CDC. By “effector cell” as used herein is meant a cellof the immune system that expresses one or more Fc receptors andmediates one or more effector functions. Effector cells include but arenot limited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys.

By “Fab” or “Fab region” as used herein is meant the polypeptides thatcomprise the V_(H), CH1, V_(H), and C_(L) immunoglobulin domains. Fabmay refer to this region in isolation, or this region in the context ofa full length antibody or antibody fragment.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat. Fc may refer to this region inisolation, or this region in the context of an Fc polypeptide, forexample an antibody. By “Fc polypeptide” as used herein is meant apolypeptide that comprises all or part of an Fc region. Fc polypeptidesinclude antibodies, Fc fusions, isolated Fcs, and Fc fragments.

By “Fc gamma receptor” or “FcγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the FcγR genes. In humans this family includesbut is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,incorporated herein it its entirety by reference), as well as anyundiscovered human FcγRs or FcγR isoforms or allotypes. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.

By “Fc ligand” or “Fc receptor” as used herein is meant a molecule,e.g., a polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to FcγRs, FcRn, C1q, C3, mannan binding lectin, mannosereceptor, staphylococcal protein A, streptococcal protein G, and viralFcγR. Fc ligands also include Fc receptor homologs (FcRH), which are afamily of Fc receptors that are homologous to the FcγRs (Davis et al.,2002, Immunological Reviews 190:123-136, incorporated herein it itsentirety by reference). Fc ligands may include undiscovered moleculesthat bind Fc.

By “IgG” as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4.In mice this class comprises IgG1, IgG2a, IgG2b, IgG3. By“immunoqlobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full length antibodies, antibody fragments, and individualimmunoglobulin domains. By “immunoqlobulin (Ig) domain” herein is meanta region of an immunoglobulin that exists as a distinct structuralentity as ascertained by one skilled in the art of protein structure. Igdomains typically have a characteristic β-sandwich folding topology. Theknown Ig domains in the IgG class of antibodies are V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

By “modification” herein is meant an alteration in the physical,chemical, or sequence properties of a protein, polypeptide, antibody, orimmunoglobulin. Modifications of the invention are amino acidmodifications and glycoform modifications.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution 1332E refers to avariant polypeptide, in this case a constant heavy chain variant, inwhich the isoleucine at position 332 is replaced with glutamic acid. TheWT residue may or may not be designated. For the preceding example, 332Eindicates the substitution of position 332 with a glutamic acid. For thepurposes herein, multiple substitutions are typically separated by aslash. For example, 239D/332E refers to a double variant comprising thesubstitutions 239D and 332E. By “amino acid insertion” or “insertion” asused herein is meant the addition of an amino acid at a particularposition in a parent polypeptide sequence. For example, insert −236Gdesignates an insertion of glycine at position 236. By “amino aciddeletion” or “deletion” as used herein is meant the removal of an aminoacid at a particular position in a parent polypeptide sequence. Forexample, G236− designates the deletion of glycine at position 236.

By “glycoform modification” or “modified glycoform” or “engineeredglycoform” as used herein is meant a carbohydrate composition that iscovalently attached to a protein, for example an antibody, wherein thecarbohydrate composition differs chemically from that of a parentprotein. Modified glycoform typically refers to the differentcarbohydrate or oligosaccharide; thus for example an antibody maycomprise a modified glycoform. Alternatively, modified glycoform mayrefer to the antibody that comprises the different carbohydrate oroligosaccharide.

By “parent polypeptide”, “parent protein”, “precursor polypeptide”, or“precursor protein” as used herein is meant a polypeptide that issubsequently modified to generate a variant, e.g., any polypeptide whichserves as a template and/or basis for at least one amino acidmodification described herein. The parent polypeptide may be a naturallyoccurring polypeptide, or a variant or engineered version of a naturallyoccurring polypeptide. Parent polypeptide may refer to the polypeptideitself, compositions that comprise the parent polypeptide, or the aminoacid sequence that encodes it. Accordingly, by “parent antibody” or“parent immunoglobulin” as used herein is meant an antibody orimmunoglobulin that is modified to generate a variant (e.g., a parentantibody may include, but is not limited to, a protein comprising theconstant region of a naturally occurring Ig).

By “protein” or “polypeptide” as used herein is meant at least twocovalently attached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The protein may be made up of naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index as in Kabat. Correspondingpositions are determined as outlined herein, generally through alignmentwith other parent sequences.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 and N297) is a residue at position 297 in thehuman antibody IgG1.

By “target antigen” or “target” or “antigen” as used herein is meant themolecule that is bound specifically by the variable region of a givenantibody. A target antigen may be a protein, carbohydrate, lipid, orother chemical compound. By “target cell” as used herein is meant a cellthat expresses a target antigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or V_(H) genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.

By “variant protein”, “protein variant”, “variant polypeptide”, or“polypeptide variant” as used herein is meant a polypeptide sequencethat differs from that of a parent polypeptide sequence by virtue of atleast one amino acid modification or at least one glycoformmodification. Variant polypeptide may refer to the polypeptide itself, acomposition comprising the polypeptide, or the amino sequence thatencodes it. In one embodiment, the variant polypeptide has at least oneamino acid modification compared to the parent polypeptide, e.g. fromabout one to about ten amino acid modifications, e.g., from about one toabout five amino acid modifications compared to the parent. The variantpolypeptide sequence herein may possess at least about 80% homology witha parent polypeptide sequence, e.g., at least about 90% homology, atleast about 95% homology, etc. Accordingly, by “variant antibody” or“antibody variant” as used herein is meant an antibody sequence thatdiffers from that of a parent antibody sequence by virtue of at leastone amino acid modification or at least one glycoform modification.Variant antibody or antibody variant may refer to the antibodypolypeptide itself, compositions comprising the antibody variantpolypeptide, or the amino acid sequence that encodes it. Accordingly, by“constant heavy chain variant” or “constant light chain variant” or “Fcvariant” as used herein is meant a constant heavy chain, constant lightchain, or Fc region polypeptide or sequence, respectively, that differsin sequence from that of a parent sequence by virtue of at least oneamino acid modification or at least one glycoform modification.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein, polypeptide, antibody, immunoglobulin, IgG,etc., has an amino acid sequence or a nucleotide sequence that has notbeen intentionally modified.

For all immunoglobulin heavy chain constant region positions discussedin the present invention, numbering is according to the EU index as inKabat (Kabat et al., 1991, Sequences of Proteins of ImmunologicalInterest, 5th Ed., United States Public Health Service, NationalInstitutes of Health, Bethesda, incorporated herein it its entirety byreference). The “EU index as in Kabat” refers to the residue numberingof the human IgG1 EU antibody, as described in Edelman et al., 1969,Biochemistry 63:78-85, incorporated herein it its entirety by reference.

Antibodies

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. The light and heavy chainvariable regions show significant sequence diversity between antibodies,and are responsible for binding the target antigen. Each chain is madeup of individual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins.

Natural antibody structural units typically comprise a tetramer. Eachtetramer is typically composed of two identical pairs of polypeptidechains, each pair having one “light” chain (typically having a molecularweight of about 25 kDa) and one “heavy” chain (typically having amolecular weight of about 50-70 kDa). Each of the light and heavy chainsare made up of two distinct regions, referred to as the variable andconstant regions. For the IgG class of immunoglobulins, the heavy chainis composed of four immunoglobulin domains linked from N- to C-terminusin the order V_(H)-CH1-CH2-CH3, referring to the heavy chain variabledomain, heavy chain constant domain 1, heavy chain constant domain 2,and heavy chain constant domain 3 respectively (also referred to asV_(H)-Cγ1-Cγ2-Cγ3, referring to the heavy chain variable domain,constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3domain respectively). The IgG light chain is composed of twoimmunoglobulin domains linked from N- to C-terminus in the orderV_(L)-C_(L), referring to the light chain variable domain and the lightchain constant domain respectively. The constant regions show lesssequence diversity, and are responsible for binding a number of naturalproteins to elicit important biochemical events.

The variable region of an antibody contains the antigen bindingdeterminants of the molecule, and thus determines the specificity of anantibody for its target antigen. The variable region is so named becauseit is the most distinct in sequence from other antibodies within thesame class. In the variable region, three loops are gathered for each ofthe V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.There are 6 CDRs total, three each per heavy and light chain, designatedV_(H) CDR1, V_(H) CDR2, V_(H) CDR3, V_(L) CDR1, V_(L) CDR2, and V_(L)CDR3. The variable region outside of the CDRs is referred to as theframework (FR) region. Although not as diverse as the CDRs, sequencevariability does occur in the FR region between different antibodies.Overall, this characteristic architecture of antibodies provides astable scaffold (the FR region) upon which substantial antigen bindingdiversity (the CDRs) can be explored by the immune system to obtainspecificity for a broad array of antigens. A number of high-resolutionstructures are available for a variety of variable region fragments fromdifferent organisms, some unbound and some in complex with antigen.Sequence and structural features of antibody variable regions aredisclosed, for example, in Morea et al., 1997, Biophys Chem 68:9-16;Morea et al., 2000, Methods 20:267-279, and the conserved features ofantibodies are disclosed, for example, in Maynard et al., 2000, Annu RevBiomed Eng 2:339-376, each incorporated herein it its entirety byreference.

Antibodies are grouped into classes, also referred to as isotypes, asdetermined genetically by the constant region. Human constant lightchains are classified as kappa (Cκ) and lambda (Cλ) light chains. Humanheavy chains are classified as mu, delta, gamma, alpha, or epsilon, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. The IgG class is the most commonly used for therapeuticpurposes. By “IgG” as used herein is meant a polypeptide belonging tothe class of antibodies that are substantially encoded by a recognizedimmunoglobulin gamma gene. In humans this class comprises subclassesIgG1, IgG2, IgG3, and IgG4. In mice this class comprises subclassesIgG1, IgG2a, IgG2b, IgG3. IgM has subclasses, including, but not limitedto, IgM1 and IgM2. IgA has several subclasses, including but not limitedto IgA1 and IgA2. Thus, “isotype” as used herein is meant any of theclasses or subclasses of immunoglobulins defined by the chemical andantigenic characteristics of their constant regions. The known humanimmunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1,IgM2, IgD, and IgE. FIG. 1 provides the sequences of the human lightchain kappa and heavy chain gamma constant chains. FIGS. 1 b & 1 c showsan alignment of the human IgG constant heavy chains.

Also useful for the invention may be IgGs that are hybrid compositionsof the natural human IgG isotypes. Effector functions such as ADCC,ADCP, CDC, and serum half-life differ significantly between thedifferent classes of antibodies, including for example human IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgG, and IgM (Michaelsen et al., 1992,Molecular Immunology, 29(3): 319-326, entirely incorporated byreference). A number of studies have explored IgG1, IgG2, IgG3, and IgG4variants in order to investigate the determinants of the effectorfunction differences between them. See for example Canfield & Morrison,1991, J. Exp. Med. 173: 1483-1491; Chappel et al., 1991, Proc. Natl.Acad. Sci. USA 88(20): 9036-9040; Chappel et al., 1993, Journal ofBiological Chemistry 268:25124-25131; Tao et al., 1991, J. Exp. Med.173: 1025-1028; Tao et al., 1993, J. Exp. Med. 178: 661-667; Redpath etal., 1998, Human Immunology, 59, 720-727, all entirely incorporated byreference.

As described in U.S. Ser. No. 11/256,060, filed Oct. 21, 2005, entitled“IgG Immunoglobulin Variants with Optimized Effector Function”, hereinexpressly incorporated by reference, it is possible to engineer aminoacid modifications in an antibody that comprise constant regions fromother immunoglobulin classes, for example as those illustrated in thealignments in FIG. 1. Such engineered hybrid IgG compositions mayprovide improved effector function properties, including improved ADCC,phagocytosis, CDC, and serum half-life. For example, as illustrated byFIG. 1, an IgG1/IgG3 hybrid variant may be constructed by substitutingIgG1 positions in the CH2 and CH3 region with the amino acids from IgG3at positions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more substitutionsselected from the group consisting of: 274Q, 276K, 300F, 339T, 356E,358M, 384S, 392N, 397M, 422I, 435R, and 436F, wherein numbering isaccording to the EU index. Such variant may provide alternate and/orimproved effector function properties.

As another example, relatively poor effector function of IgG2 may beimproved by replacing key FcγR binding residues with the correspondingamino acids in an IgG with better effector function. For example, keyresidue differences between IgG2 and IgG1 with respect to FcγR bindingmay include P233, V234, A235, −236 (referring to a deletion in IgG2relative to IgG1), and G327. Thus one or more amino acid modificationsin the parent IgG2 wherein one or more of these residues is replacedwith the corresponding IgG1 amino acids, P233E, V234L, A235L, -236G(referring to an insertion of a glycine at position 236), and G327A, mayprovide enhanced effector function. The sequence of such an IgG,comprising a hybrid of residues from IgG1 and IgG2, referred to hereinas “Hybrid”, is provided in FIG. 1.

As is well known in the art, immunoglobulin polymorphisms exist in thehuman population. Gm polymorphism is determined by the IGHG1, IGHG2 andIGHG3 genes which have alleles encoding allotypic antigenic determinantsreferred to as G1m, G2m, and G3m allotypes for markers of the humanIgG1, IgG2 and IgG3 molecules (no Gm allotypes have been found on thegamma 4 chain). Markers may be classified into ‘allotypes’ and‘isoallotypes’. These are distinguished on different serological basesdependent upon the strong sequence homologies between isotypes.Allotypes are antigenic determinants specified by allelic forms of theIg genes. Allotypes represent slight differences in the amino acidsequences of heavy or light chains of different individuals. Even asingle amino acid difference can give rise to an allotypic determinant,although in many cases there are several amino acid substitutions thathave occurred. Allotypes are sequence differences between alleles of asubclass whereby the antisera recognize only the allelic differences. Anisoallotype is an allele in one isotype which produces an epitope whichis shared with a non-polymorphic homologous region of one or more otherisotypes and because of this the antisera will react with both therelevant allotypes and the relevant homologous isotypes (Clark, 1997,IgG effector mechanisms, Chem. Immunol. 65:88-110; Gorman & Clark, 1990,Semin. Immunol. 2(6):457-66, each incorporated herein it its entirety byreference).

Allelic forms of human immunoglobulins have been well-characterized (WHOReview of the notation for the allotypic and related markers of humanimmunoglobulins. J Immunogen 1976, 3: 357-362; WHO Review of thenotation for the allotypic and related markers of human immunoglobulins.1976, Eur. J. Immunol. 6, 599-601; E. van Loghem, 1986, Allotypicmarkers, Monogr Allergy 19: 40-51, each incorporated herein it itsentirety by reference). Additionally, other polymorphisms have beencharacterized (Kim, et al., 2001, J. Mol. Evol. 54:1-9, incorporatedherein it its entirety by reference). At present, 18 Gm allotypes areknown: G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m(5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b5,b0, b3, b4, s, t, g1, c5, u, v, g5) (Lefranc, et al., The human IgGsubclasses: molecular analysis of structure, function and regulation.Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum.Genet.: 50, 199-211, each incorporated herein it its entirety byreference). Allotypes that are inherited in fixed combinations arecalled Gm haplotypes. FIG. 2 shows common haplotypes of the gamma chainof human IgG1 (FIG. 2 a) and IgG2 (FIG. 2 b) showing the positions andthe relevant amino acid substitutions. The antibodies of the presentinvention may be substantially encoded by any allotype, isoallotype, orhaplotype of any immunoglobulin gene.

Antibodies of the present invention may be substantially encoded bygenes from any organism, e.g., mammals, including but not limited tohumans, rodents including but not limited to mice and rats, lagomorphaincluding but not limited to rabbits and hares, camelidae including butnot limited to camels, llamas, and dromedaries, and non-human primates,including but not limited to Prosimians, Platyrrhini (New Worldmonkeys), Cercopithecoidea (Old World monkeys), and Hominoidea includingthe Gibbons and Lesser and Great Apes. In one embodiment, the antibodiesof the present invention are substantially human. The antibodies of thepresent invention may be substantially encoded by immunoglobulin genesbelonging to any of the antibody classes. In one embodiment, theantibodies of the present invention comprise sequences belonging to theIgG class of antibodies, including human subclasses IgG1, IgG2, IgG3,and IgG4. In an alternate embodiment, the antibodies of the presentinvention comprise sequences belonging to the IgA (including humansubclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes of antibodies.The antibodies of the present invention may comprise more than oneprotein chain. That is, the present invention may find use in anantibody that is a monomer or an oligomer, including a homo- orhetero-oligomer.

In one embodiment, the antibodies of the invention are based on humanIgG sequences, and thus human IgG sequences are used as the “base”sequences against which other sequences are compared, including but notlimited to sequences from other organisms, for example rodent andprimate sequences, as well as sequences from other immunoglobulinclasses such as IgA, IgE, IgD, IgM, and the like. It is contemplatedthat, although the antibodies of the present invention are engineered inthe context of one parent antibody, the variants may be engineered in or“transferred” to the context of another, second parent antibody. This isdone by determining the “equivalent” or “corresponding” residues andsubstitutions between the first and second antibodies, typically basedon sequence or structural homology between the sequences of the twoantibodies. In order to establish homology, the amino acid sequence of afirst antibody outlined herein is directly compared to the sequence of asecond antibody. After aligning the sequences, using one or more of thehomology alignment programs known in the art (for example usingconserved residues as between species), allowing for necessaryinsertions and deletions in order to maintain alignment (i.e., avoidingthe elimination of conserved residues through arbitrary deletion andinsertion), the residues equivalent to particular amino acids in theprimary sequence of the first antibody are defined. Alignment ofconserved residues may conserve 100% of such residues. However,alignment of greater than 75% or as little as 50% of conserved residuesis also adequate to define equivalent residues. Equivalent residues mayalso be defined by determining structural homology between a first andsecond antibody that is at the level of tertiary structure forantibodies whose structures have been determined. In this case,equivalent residues are defined as those for which the atomiccoordinates of two or more of the main chain atoms of a particular aminoacid residue of the parent or precursor (N on N, CA on CA, C on C and Oon O) are within 0.13 nm, e.g., 0.1 nm, after alignment. Alignment isachieved after the best model has been oriented and positioned to givethe maximum overlap of atomic coordinates of non-hydrogen protein atomsof the proteins. Regardless of how equivalent or corresponding residuesare determined, and regardless of the identity of the parent antibody inwhich the antibodies are made, what is meant to be conveyed is that theantibodies discovered by the present invention may be engineered intoany second parent antibody that has significant sequence or structuralhomology with the antibody. Thus for example, if a variant antibody isgenerated wherein the parent antibody is human IgG1, by using themethods described above or other methods for determining equivalentresidues, the variant antibody may be engineered in a human IgG2 parentantibody, a human IgA parent antibody, a mouse IgG2a or IgG2b parentantibody, and the like. Again, as described above, the context of theparent antibody does not affect the ability to transfer the antibodiesof the present invention to other parent antibodies. For example, thevariant antibodies that are engineered in a human IgG1 antibody thattargets one antigen epitope may be transferred into a human IgG2antibody that targets a different antigen epitope, and so forth.

In the IgG class of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the domains of theconstant heavy chain, including, the constant heavy (CH) domains and thehinge. In the context of IgG antibodies, the IgG isotypes each havethree CH regions: “CH1” refers to positions 118-220, “CH2” refers topositions 237-340, and “CH3” refers to positions 341-447 according tothe EU index as in Kabat. By “hinge” or “hinge region” or “antibodyhinge region” or “immunoglobulin hinge region” herein is meant theflexible polypeptide comprising the amino acids between the first andsecond constant domains of an antibody. Structurally, the IgG CH1 domainends at EU position 220, and the IgG CH2 domain begins at residue EUposition 237. Thus for IgG the hinge is herein defined to includepositions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein thenumbering is according to the EU index as in Kabat. In some embodiments,for example in the context of an Fc region, the lower hinge is included,with the “lower hinge” generally referring to positions 226 or 230. Theconstant heavy chain, as defined herein, refers to the N-terminus of theCH1 domain to the C-terminus of the CH3 domain, thus comprisingpositions 118-447, wherein numbering is according to the EU index. Theconstant light chain comprises a single domain, and as defined hereinrefers to positions 108-214 of Cκ or Cλ, wherein numbering is accordingto the EU index.

Specifically included within the definition of “antibody” arefull-length antibodies. By “full length antibody” herein is meant thestructure that constitutes the natural biological form of an antibody,including variable and constant regions. For example, in most mammals,including humans and mice, the full length antibody of the IgG class isa tetramer and consists of two identical pairs of two immunoglobulinchains, each pair having one light and one heavy chain, each light chaincomprising immunoglobulin domains V_(L) and C_(L), and each heavy chaincomprising immunoglobulin domains V_(H), CH1 (Cγ1), CH2 (Cγ2), and CH3(Cγ3). In some mammals, for example in camels and llamas, IgG antibodiesmay consist of only two heavy chains, each heavy chain comprising avariable domain attached to the Fc region.

Alternatively, the antibodies can be a variety of structures, including,but not limited to antibody fragments. Antibody fragments include butare not limited to bispecific antibodies, minibodies, domain antibodies,synthetic antibodies, antibody mimetics, chimeric antibodies, antibodyfusions (sometimes referred to as “antibody conjugates”), and fragmentsof each, respectively. Specific antibody fragments include, but are notlimited to, (i) the Fab fragment consisting of VL, VH, CL and CH1domains, (ii) the Fd fragment consisting of the VH and CH1 domains,(iii) the Fv fragment consisting of the VL and VH domains of a singleantibody; (iv) the dAb fragment, which consists of a single variableregion, (v) isolated CDR regions, (vi) F(ab′)2 fragments, a bivalentfragment comprising two linked Fab fragments (vii) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (viii) bispecific single chain Fv dimers and (ix)“diabodies” or “triabodies”, multivalent or multispecific fragmentsconstructed by gene fusion. The antibody fragments may be modified. Forexample, the molecules may be stabilized by the incorporation ofdisulfide bridges linking the VH and VL domains. Examples of antibodyformats and architectures are described in Holliger & Hudson, 2006,Nature Biotechnology 23(9):1126-1136, and Carter 2006, Nature ReviewsImmunology 6:343-357 and references cited therein, all expresslyincorporated by reference.

Antibodies of the invention may include multispecific antibodies,notably bispecific antibodies, also sometimes referred to as“diabodies”. These are antibodies that bind to two (or more) differentantigens. Diabodies can be manufactured in a variety of ways known inthe art, e.g., prepared chemically or from hybrid hybridomas. In oneembodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. In somecases, the scFv can be joined to the Fc region, and may include some orall of the hinge region. For a description of multispecific antibodiessee Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136 andreferences cited therein, all expressly incorporated by reference.

In one embodiment, the antibody of the invention is an antibodyfragment. Of particular interest are antibodies that comprise Fcregions, Fc fusions, and the constant region of the heavy chain(CH1-hinge-CH2-CH3). Antibodies of the present invention may comprise Fcfragments. An Fc fragment of the present invention may comprise from1-90% of the Fc region, e.g., 10-90%, 30-90%, etc. Thus for example, anFc fragment of the present invention may comprise an IgG1 Cγ2 domain, anIgG1 Cγ2 domain and hinge region, an IgG1 Cγ3 domain, and so forth. Inone embodiment, an Fc fragment of the present invention additionallycomprises a fusion partner, effectively making it an Fc fragment fusion.Fc fragments may or may not contain extra polypeptide sequence.

Chimeric, Humanized, and Fully Human Antibodies

Immunogenicity is the result of a complex series of responses to asubstance that is perceived as foreign, and may include production ofneutralizing and non-neutralizing antibodies, formation of immunecomplexes, complement activation, mast cell activation, inflammation,hypersensitivity responses, and anaphylaxis. Several factors cancontribute to protein immunogenicity, including but not limited toprotein sequence, route and frequency of administration, and patientpopulation. Immunogenicity may limit the efficacy and safety of aprotein therapeutic in multiple ways. Efficacy can be reduced directlyby the formation of neutralizing antibodies. Efficacy may also bereduced indirectly, as binding to either neutralizing ornon-neutralizing antibodies typically leads to rapid clearance fromserum. Severe side effects and even death may occur when an immunereaction is raised. Thus in one embodiment, protein engineering is usedto reduce the immunogenicity of the antibodies of the present invention.

In some embodiments, the scaffold components can be a mixture fromdifferent species. Such antibody may be a chimeric antibody and/or ahumanized antibody. In general, both “chimeric antibodies” and“humanized antibodies” refer to antibodies that combine regions frommore than one species. “Chimeric antibodies” traditionally comprisevariable region(s) from a mouse (or rat, in some cases) and the constantregion(s) from a human (Morrison et al., 1984, Proc Natl Acad Sci USA81: 6851-6855, incorporated herein it its entirety by reference).

By “humanized” antibody as used herein is meant an antibody comprising ahuman framework region (FR) and one or more complementarity determiningregions (CDRs) from a non-human (usually mouse or rat) antibody. Thenon-human antibody providing the CDRs is called the “donor” and thehuman immunoglobulin providing the framework is called the “acceptor”.Humanization relies principally on the grafting of donor CDRs ontoacceptor (human) VL and VH frameworks (Winter U.S. Pat. No. 5,225,539,incorporated herein it its entirety by reference). This strategy isreferred to as “CDR grafting”. “Backmutation” of selected acceptorframework residues to the corresponding donor residues is often requiredto regain affinity that is lost in the initial grafted construct (U.S.Pat. No. 5,693,762, incorporated herein it its entirety by reference).The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region. Avariety of techniques and methods for humanizing and reshaping non-humanantibodies are well known in the art (See Tsurushita & Vasquez, 2004,Humanization of Monoclonal Antibodies, Molecular Biology of B Cells,533-545, Elsevier Science (USA), and references cited therein, eachincorporated herein it its entirety by reference). Humanization or othermethods of reducing the immunogenicity of nonhuman antibody variableregions may include resurfacing methods, as described for example inRoguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973,incorporated herein it its entirety by reference. In one embodiment,selection based methods may be employed to humanize and/or affinitymature antibody variable regions, that is, to increase the affinity ofthe variable region for its target antigen. Other humanization methodsmay involve the grafting of only parts of the CDRs, including but notlimited to methods described in U.S. Ser. No. 09/810,502; Tan et al.,2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol.169:3076-3084, incorporated herein it its entirety by reference.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 10/153,159 andrelated applications, each incorporated herein it its entirety byreference.

In certain variations, the immunogenicity of the antibody is reducedusing a method described in U.S. Ser. No. 11/004,590, entitled “Methodsof Generating Variant Proteins with Increased Host String Content andCompositions Thereof”, filed on Dec. 3, 2004, incorporated herein it itsentirety by reference.

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an antibody ofthe present invention. See for example U.S. Ser. No. 09/903,378, U.S.Ser. No. 10/754,296, U.S. Ser. No. 11/249,692, and references citedtherein, all expressly incorporated by reference.

In an alternate embodiment, the antibodies of the present invention maybe fully human, that is the sequences of the antibodies are completelyor substantially human. “Fully human antibody” or “complete humanantibody” refers to a human antibody having the gene sequence of anantibody derived from a human chromosome with the modifications outlinedherein. A number of methods are known in the art for generating fullyhuman antibodies, including the use of transgenic mice (Bruggemann etal., 1997, Curr Opin Biotechnol 8:455-458,) or human antibody librariescoupled with selection methods (Griffiths et al., 1998, Curr OpinBiotechnol 9:102-108), each incorporated herein it its entirety byreference.

Antibodies that Target CD40

The antibodies of the present invention may be virtually any antibodythat binds to CD40, e.g., may comprise the variable regions (e.g., theCDRs) of any known or undiscovered anti-CD40 antibody. Antibodies of theinvention may display selectivity for CD40. Examples include full-lengthversus splice variants, cell-surface vs. soluble forms, selectivity forvarious polymorphic variants, or selectivity for specific conformationalforms of a target. An antibody of the present invention may bind anyepitope or region on CD40 and may be specific for fragments, mutantforms, splice forms, or aberrant forms of the antigens. A number ofuseful antibodies have been discovered that target CD40 that may finduse in the present invention.

Suitable CD40 antibodies or immunoadhesins include the CD40 antibodiesor immunoadhesins S2C6 (Paulie et al., Cancer Immunol. Immunother., Vol17, No 3, (1984) pp. 173-179), SGN-14 (chimeric S2C6; U.S. Pat. No.6,843,989), CHIR-12.12 (US Pub. No. 2007/0218060, filed Nov. 4, 2004),5D12 (U.S. Pat. No. 5,874,082; de Boer M. et al., 1992. J ImmunolMethods. 152:15-23), 3A8 (ATCC cat#HB-12024-hybridoma), and G28-5 (ATCCcat#HB-9110 hybridoma; Clark E. A. et al., 1988. Eur J Immunol.18:451-457).

The antibodies of the present invention may find use in a wide range ofproducts. In one embodiment the antibody of the invention is atherapeutic, a diagnostic, or a research reagent. In one embodiment, anantibody of the invention is a therapeutic. Alternatively, the antibodyof the present invention may be used for agricultural or industrialuses. An antibody of the present invention may find use in an antibodycomposition that is monoclonal or polyclonal. The antibodies of thepresent invention may be agonists, antagonists, neutralizing,inhibitory, or stimulatory. In one embodiment, the antibodies of thepresent invention are used to kill target cells that bear the targetantigen, for example cancer cells. In an alternate embodiment, theantibodies of the present invention are used to block, antagonize, oragonize the target antigen. In an alternate embodiment, the antibodiesof the present invention are used to block, antagonize, or agonize thetarget antigen and kill the target cells that bear the target antigen.

It will be recognized that the sequences identified as S2C6 VH (H0-H4)and S2C6 VL (L0-L3) can be combined in any combination in an antibody.Further, these sequences may be independently modified by adding all orpart of an Fc region or Fc variant as disclosed herein. The modifiedsequences can also be combined in any combination in an antibody.

Modifications for Optimizing Effector Function

The present invention is directed to antibodies comprisingmodifications, wherein the modifications alter affinity to one or moreFc receptors, and/or alter the ability of the antibody to mediate one ormore effector functions. Modifications of the invention include aminoacid modifications and glycoform modifications.

Amino Acid Modifications

As described in U.S. Ser. No. 11/124,620, filed May 5, 2005, entitled“Optimized Fc Variants”, and incorporated herein it its entirety byreference, amino acid modifications at heavy chain constant regionpositions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 255,258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, and 337, allow modification of FcγR bindingproperties, effector function, and potentially clinical properties ofantibodies.

In particular, variants that alter binding to one or more human Fcreceptors may comprise an amino acid modification in the heavy chainconstant region, as described herein, selected from the group consistingof 221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W,227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y,231E, 231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F,233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V,233W, 233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N,234P, 234Q, 234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F,235G, 235H, 235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V,235W, 235Y, 236A, 236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N,236P, 236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H,237I, 237K, 237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W,237Y, 238D, 238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q,238R, 238S, 238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I,239K, 239L, 239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240A,240I, 240M, 240T, 241D, 241E, 241L, 241R, 241S, 241W, 241Y, 243E, 243H,243L, 243Q, 243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G,247V, 249H, 249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 260H,260Y, 262A, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 264A, 264D,264E, 264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N, 264P, 264Q, 264R,264S, 264T, 264W, 264Y, 265F, 265G, 265H, 265I, 265K, 265L, 265M, 265N,265P, 265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I, 266M, 266T,267D, 267E, 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P, 267Q, 267R,267T, 267V, 267W, 267Y, 268D, 268E, 268F, 268G, 268I, 268K, 268L, 268M,268P, 268Q, 268R, 268T, 268V, 268W, 269F, 269G, 269H, 269I, 269K, 269L,269M, 269N, 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270G, 270H,270I, 270L, 270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y, 271A, 271D,271E, 271F, 271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S,271T, 271V, 271W, 271Y, 272D, 272F, 272G, 272H, 272I, 272K, 272L, 272M,272P, 272R, 272S, 272T, 272V, 272W, 272Y, 273I, 274D, 274E, 274F, 274G,274H, 274I, 274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L,275W, 276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P, 276R, 276S,276T, 276V, 276W, 276Y, 278D, 278E, 278G, 278H, 278I, 278K, 278L, 278M,278N, 278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P,280W, 281D, 281E, 281K, 281N, 281P, 281Q, 281Y, 282E, 282G, 282K, 282P,282Y, 283G, 283H, 283K, 283L, 283P, 283R, 283Y, 284D, 284E, 284L, 284N,284Q, 284T, 284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286Q, 286P,286Y, 288D, 288E, 288Y, 290D, 290H, 290L, 290N, 290W, 291D, 291E, 291G,291H, 291I, 291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I,293L, 293M, 293N, 293P, 293R, 293S, 293T, 293V, 293W, 293Y, 294F, 294G,294H, 294I, 294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y,295D, 295E, 295F, 295G, 295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T,295V, 295W, 295Y, 296A, 296D, 296E, 296G, 296H, 296I, 296K, 296L, 296M,296N, 296Q, 296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G, 297H, 297I,297K, 297L, 297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 298A,298D, 298E, 298F, 298H, 298I, 298K, 298M, 298N, 298Q, 298R, 298T, 298W,298Y, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N,299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H,300K, 300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E,301H, 301Y, 302I, 303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E,305T, 305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F,320G, 320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D,322F, 322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y, 323I, 324D,324F, 324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y,325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q,325R, 325S, 325T, 325V, 325W, 325Y, 326E, 326I, 326L, 326P, 326T, 327D,327E, 327F, 327H, 327I, 327K, 327L, 327M, 327N, 327P, 327R, 327S, 327T,327V, 327W, 327Y, 328A, 328D, 328E, 328F, 328G, 328H, 328I, 328K, 328M,328N, 328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F,329G, 329H, 329I, 329K, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V,329W, 329Y, 330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R,330S, 330T, 330V, 330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q,331R, 331T, 331V, 331W, 331Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L,332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 333A, 333F,333H, 333I, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 334I, 334L, 334P,334T, 335D, 335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S,335V, 335W, 335Y, 336E, 336K, 336Y, 337E, 337H, and 337N, whereinnumbering is according to the EU index.

As described in U.S. Ser. No. 11/090,981, filed Mar. 24, 2005, entitled“Immunoglobulin variants outside the Fc region”, and incorporated hereinit its entirety by reference, amino acid modifications at heavy chainconstant region positions 118, 119, 120, 121, 122, 124, 126, 129, 131,132, 133, 135, 136, 137, 138, 139, 147, 148, 150, 151, 152, 153, 155,157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 183, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 201, 203, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 216, 217, 218, 219, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, and 236, allowmodification of FcγR binding properties, effector function, andpotentially clinical properties of antibodies.

As described in U.S. Ser. No. 11/090,981, filed Mar. 24, 2005, entitled“Immunoglobulin variants outside the Fc region”, and incorporated hereinit its entirety by reference, amino acid modifications at light chainconstant region positions 108, 109, 110, 111, 112, 114, 116, 121, 122,123, 124, 125, 126, 127, 128, 129, 131, 137, 138, 140, 141, 142, 143,145, 147, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 176,180, 181, 182, 183, 184, 185, 187, 188, 189, 190, 191, 193, 195, 197,199, 200, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, allowmodification of FcγR binding properties, effector function, andpotentially clinical properties of antibodies.

In particular, variants that alter binding to one or more human Fcreceptors may comprise an amino acid modification in the heavy chainconstant region, as described herein, selected from the group consistingof 118K, 118E, 118Y, 119R, 119E, 119Y, 120R, 120E, 120I, 121E, 121Y,121H, 122E, 122R, 124K, 124E, 124Y, 126K, 126D, 129L, 129D, 131G, 131T,132D, 132R, 132L, 133R, 133E, 133L, 135I, 135E, 135K, 136E, 136K, 136I,137E, 138S, 138R, 138D, 139I, 139E, 139K, 147A, 147E, 148Y, 148K, 150L,150K, 150E, 151A, 151D, 152L, 152K, 153L, 153D, 155E, 155K, 155I, 157E,157K, 157Y, 159K, 159D, 159L, 160K, 160E, 160Y, 161D, 162D, 162K, 162Y,163R, 164R, 164E, 164Y, 165D, 165R, 165Y, 166D, 167A, 168L, 169E, 171G,171H, 172K, 172L, 172E, 173T, 173D, 174E, 174K, 174Y, 175D, 175L, 176D,176R, 176L, 177R, 177E, 177Y, 178D, 179K, 179Y, 179E, 180K, 180L, 180E,183T, 187I, 187K, 187E, 188I, 189D, 189G, 190I, 190K, 190E, 191D, 191R,191Y, 192N, 192R, 192L, 193F, 193E, 194R, 194D, 195R, 195D, 195Y, 196K,196D, 196L, 197R, 197E, 197Y, 198L, 199T, 199D, 199K, 201E, 201K, 201L,203D, 203L, 203K, 205D, 205L, 206A, 206E, 207K, 207D, 208R, 208E, 208Y,209E, 209K, 209Y, 210L, 210E, 210Y, 211R, 211E, 211Y, 212Q, 212K, 212H,212L, 212Y, 213N, 213E, 213H, 213L, 213Y, 214N, 214E, 214H, 214L, 214Y,216N, 216K, 216H, 216L, 216Y, 217D, 217H, 217A, 217V, 217G, 218D, 218E,218Q, 218T, 218H, 218L, 218Y, 219D, 219E, 219Q, 219K, 219T, 219H, 219L,219I, 219Y, 205A, 210A, 213A, 214A, 218A, 221K, 221Y, 221E, 221N, 221Q,221R, 221S, 221T, 221H, 221A, 221V, 221L, 221I, 221F, 221M, 221W, 221P,221G, 222E, 222Y, 222D, 222N, 222Q, 222R, 222S, 222T, 222H, 222V, 222L,222I, 222F, 222M, 222W, 222P, 222G, 222A, 223D, 223N, 223Q, 223R, 223S,223H, 223A, 223V, 223L, 223I, 223F, 223M, 223Y, 223W, 223P, 223G, 223E,223K, 224D, 224N, 224Q, 224K, 224R, 224S, 224T, 224V, 224L, 224I, 224F,224M, 224W, 224P, 224G, 224E, 224Y, 224A, 225D, 225N, 225Q, 225R, 225S,225H, 225A, 225V, 225L, 225I, 225F, 225M, 225Y, 225P, 225G, 225E, 225K,225W, 226S, 227E, 227K, 227Y, 227G, 227D, 227N, 227Q, 227R, 227S, 227T,227H, 227A, 227V, 227L, 227I, 227F, 227M, 227W, 228K, 228Y, 228G, 228D,228N, 228Q, 228R, 228T, 228H, 228A, 228V, 228L, 228I, 228F, 228M, 228W,229S, 230A, 230E, 230Y, 230G, 230D, 230N, 230Q, 230K, 230R, 230S, 230T,230H, 230V, 230L, 230I, 230F, 230M, 230W, 231K, 231P, 231D, 231N, 231Q,231R, 231S, 231T, 231H, 231V, 231L, 231I, 231F, 231M, 231W, 232E, 232K,232Y, 232G, 232D, 232N, 232Q, 232R, 232S, 232T, 232H, 232A, 232V, 232L,232I, 232F, 232M, 232W, 233D, 233N, 233Q, 233R, 233S, 233T, 233H, 233A,233V, 233L, 233I, 233F, 233M, 233Y, 233W, 233G, 234D, 234E, 234N, 234Q,234T, 234H, 234Y, 234I, 234V, 234F, 234K, 234R, 234S, 234A, 234M, 234G,235D, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 235E, 235K,235R, 235A, 235M, 235W, 235P, 235G, 236D, 236E, 236N, 236Q, 236K, 236R,236S, 236T, 236H, 236A, 236V, 236L, 236I, 236F, 236M, 236Y, 236W, and236P, wherein numbering is according to the EU index.

In particular, variants that alter binding to one or more human Fcreceptors may comprise an amino acid modification in the light chainconstant region, as described herein, selected from the group consistingof 108D, 108I, 108Q, 109D, 109P, 109R, 110E, 110I, 110K, 111E, 111K,111L, 112E, 112R, 112Y, 114D, 114I, 114K, 116T, 121D, 122R, 122S, 122Y,123L, 123R, 124E, 125E, 125K, 126D, 126L, 126Q, 127A, 127D, 127K, 128N,129E, 129I, 129K, 131T, 137K, 137S, 138D, 138K, 138L, 140E, 140H, 140K,141E, 141K, 142D, 142G, 142L, 143A, 143L, 143R, 145D, 145T, 145Y, 147A,147E, 147K, 149D, 149Y, 150A, 151I, 151K, 152L, 152R, 152S, 153D, 153H,153S, 154E, 154R, 154V, 155E, 155I, 155K, 156A, 156D, 156R, 157N, 158D,158L, 158R, 159E, 159K, 159L, 160K, 160V, 161K, 161L, 162T, 163E, 163K,163T, 164Q, 165K, 165P, 165Y, 166E, 166M, 166S, 167K, 167L, 168K, 168Q,168Y, 169D, 169H, 169S, 170I, 170N, 170R, 171A, 171N, 171V, 172E, 172I,172K, 173K, 173L, 173Q, 174A, 176T, 180E, 180K, 180S, 181K, 182E, 182R,182T, 183D, 183L, 183P, 184E, 184K, 184Y, 185I, 185Q, 185R, 187K, 187Y,188E, 188S, 188Y, 189D, 189K, 189Y, 190E, 190L, 190R, 191E, 191R, 191S,193E, 193K, 193S, 195I, 195K, 195Q, 197E, 197K, 197L, 199E, 199K, 199Y,200S, 202D, 202R, 202Y, 203D, 203L, 203R, 204T, 205E, 205K, 206E, 206I,206K, 207A, 207E, 207L, 208E, 208K, 208T, 210A, 210E, 210K, 211A, 211E,211P, 212E, 212K, 212T, 213L, 213R, wherein numbering is according tothe EU index.

Additional substitutions that may also be used in the present inventioninclude other substitutions that modulate Fc receptor affinity,FcγR-mediated effector function, and/or complement mediated effectorfunction include but are not limited to 298A, 298T, 326A, 326D, 326E,326W, 326Y, 333A, 333S, 334L, and 334A (U.S. Pat. No. 6,737,056; Shieldset al, Journal of Biological Chemistry, 2001, 276(9):6591-6604; U.S.Pat. No. 6,528,624; Idusogie et al., 2001, J. Immunology 166:2571-2572),247L, 255L, 270E, 392T, 396L, and 421K (U.S. Ser. No. 10/754,922; U.S.Ser. No. 10/902,588), and 280H, 280Q, and 280Y (U.S. Ser. No.10/370,749), each incorporated herein it its entirety by reference.

In other embodiments, antibodies of the present invention may becombined with constant heavy chain variants that alter FcRn binding.These include modifications that modify FcRn affinity in a pH-specificmanner. In particular, variants that increase Fc binding to FcRn includebut are not limited to: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton etal., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journalof Immunology 176:346-356, U.S. Ser. No. 11/102,621, PCT/US2003/033037,PCT/US2004/011213, U.S. Ser. No. 10/822,300, U.S. Ser. No. 10/687,118,PCT/US2004/034440, U.S. Ser. No. 10/966,673, each incorporated herein itits entirety by reference), 256A, 272A, 286A, 305A, 307A, 311A, 312A,376A, 378Q, 380A, 382A, 434A (Shields et al, Journal of BiologicalChemistry, 2001, 276(9):6591-6604, U.S. Ser. No. 10/982,470, U.S. Pat.No. 6,737,056, U.S. Ser. No. 11/429,793, U.S. Ser. No. 11/429,786,PCT/US2005/029511, U.S. Ser. No. 11/208,422, each incorporated herein itits entirety by reference), 252F, 252T, 252Y, 252W, 254T, 256S, 256R,256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 433I, 433P, 433Q, 434H,434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dall Acquaet al. Journal of Immunology, 2002, 169:5171-5180, U.S. Pat. No.7,083,784, PCT/US97/03321, U.S. Pat. No. 6,821,505, PCT/US01/48432, U.S.Ser. No. 11/397,328, each incorporated herein it its entirety byreference), 257C, 257M, 257L, 257N, 257Y, 279E, 279Q, 279Y, insertion ofSer after 281, 283F, 284E, 306Y, 307V, 308F, 308Y 311V, 385H, 385N,(PCT/US2005/041220, U.S. Ser. No. 11/274,065, U.S. Ser. No. 11/436,266,each incorporated herein it its entirety by reference) 204D, 284E, 285E,286D, and 290E (PCT/US2004/037929 incorporated herein it its entirety byreference).

In some embodiments of the invention, antibodies may comprise isotypicmodifications, that is modifications in a parent IgG to the amino acidtype in an alternate IgG. For example as illustrated in FIG. 1, anIgG1/IgG3 hybrid variant may be constructed by substituting IgG1positions in the CH2 and/or CH3 region with the amino acids from IgG3 atpositions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more substitutionsselected from the group consisting of: 274Q, 276K, 300F, 339T, 356E,358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments ofthe invention, an IgG1/IgG2 hybrid variant may be constructed bysubstituting IgG2 positions in the CH2 and/or CH3 region with aminoacids from IgG1 at positions where the two isotypes differ. Thus ahybrid variant IgG antibody may be constructed that comprises one ormore modifications selected from the group consisting of 233E, 234L,235L, −236G (referring to an insertion of a glycine at position 236),and 327A.

Means for Optimizing Effector Function

The present invention is directed to antibodies comprising means foraltering affinity to one or more Fc receptors, and/or alter the abilityof the antibody to mediate one or more effector functions. Means of theinvention include amino acid modifications (e.g., positional means foroptimizing effector function, substitutional means for optimizingeffector function, etc.) and glycoform modifications (e.g., means forglycoform modifications).

Amino Acid Modifications

As described in U.S. Ser. No. 11/124,620, filed May 5, 2005, entitled“Optimized Fc Variants”, and incorporated herein it its entirety byreference, positional means for optimizing effector function include butis not limited to, modification of an amino acid at one or more heavychain constant region positions (e.g., at positions 221, 222, 223, 224,225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281,282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320, 322, 323,324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and337) which allow modification of FcγR binding properties, effectorfunction, and potentially clinical properties of antibodies.

In particular, substitutional means for optimizing effector functions,for altering binding to one or more human Fc receptors, include, but isnot limited to, a substitution of an amino acid at one or more heavychain constant region positions, e.g., one or more of the followingamino acid substitutions in the heavy chain constant region positions:221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E,227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E,231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G,233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W,233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P,234Q, 234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F, 235G,235H, 235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W,235Y, 236A, 236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P,236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I,237K, 237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y,238D, 238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R,238S, 238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K,239L, 239M, 239N, 239P, 239Q, 239R, 239T, 239V, 239W, 239Y, 240A, 240I,240M, 240T, 241D, 241E, 241L, 241R, 241S, 241W, 241Y, 243E, 243H, 243L,243Q, 243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V,249H, 249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 260H, 260Y,262A, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 264A, 264D, 264E,264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N, 264P, 264Q, 264R, 264S,264T, 264W, 264Y, 265F, 265G, 265H, 265I, 265K, 265L, 265M, 265N, 265P,265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 266I, 266M, 266T, 267D,267E, 267F, 267H, 267I, 267K, 267L, 267M, 267N, 267P, 267Q, 267R, 267T,267V, 267W, 267Y, 268D, 268E, 268F, 268G, 268I, 268K, 268L, 268M, 268P,268Q, 268R, 268T, 268V, 268W, 269F, 269G, 269H, 269I, 269K, 269L, 269M,269N, 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270G, 270H, 270I,270L, 270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y, 271A, 271D, 271E,271F, 271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T,271V, 271W, 271Y, 272D, 272F, 272G, 272H, 272I, 272K, 272L, 272M, 272P,272R, 272S, 272T, 272V, 272W, 272Y, 273I, 274D, 274E, 274F, 274G, 274H,274I, 274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L, 275W,276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P, 276R, 276S, 276T,276V, 276W, 276Y, 278D, 278E, 278G, 278H, 278I, 278K, 278L, 278M, 278N,278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P, 280W,281D, 281E, 281K, 281N, 281P, 281Q, 281Y, 282E, 282G, 282K, 282P, 282Y,283G, 283H, 283K, 283L, 283P, 283R, 283Y, 284D, 284E, 284L, 284N, 284Q,284T, 284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y,288D, 288E, 288Y, 290D, 290H, 290L, 290N, 290W, 291D, 291E, 291G, 291H,291I, 291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L,293M, 293N, 293P, 293R, 293S, 293T, 293V, 293W, 293Y, 294F, 294G, 294H,294I, 294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D,295E, 295F, 295G, 295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V,295W, 295Y, 296A, 296D, 296E, 296G, 296H, 296I, 296K, 296L, 296M, 296N,296Q, 296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G, 297H, 297I, 297K,297L, 297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 298A, 298D,298E, 298F, 298H, 298I, 298K, 298M, 298N, 298Q, 298R, 298T, 298W, 298Y,299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N, 299P,299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H, 300K,300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E, 301H,301Y, 302I, 303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E, 305T,305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F, 320G,320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F,322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y, 323I, 324D, 324F,324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A,325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R,325S, 325T, 325V, 325W, 325Y, 326E, 326I, 326L, 326P, 326T, 327D, 327E,327F, 327H, 327I, 327K, 327L, 327M, 327N, 327P, 327R, 327S, 327T, 327V,327W, 327Y, 328A, 328D, 328E, 328F, 328G, 328H, 328I, 328K, 328M, 328N,328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F, 329G,329H, 329I, 329K, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329W,329Y, 330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R, 330S,330T, 330V, 330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q, 331R,331T, 331V, 331W, 331Y, 332A, 332D, 332E, 332F, 332H, 332K, 332L, 332M,332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 333A, 333F, 333H,333I, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 334I, 334L, 334P, 334T,335D, 335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S, 335V,335W, 335Y, 336E, 336K, 336Y, 337E, 337H, and 337N, wherein numbering isaccording to the EU index.

As described in U.S. Ser. No. 11/090,981, filed Mar. 24, 2005, entitled“Immunoglobulin variants outside the Fc region”, and incorporated hereinit its entirety by reference, positional means for optimizing effectorfunction include, but is not limited to, modification of an amino acidat one or more heavy chain constant region positions (e.g., at positions118, 119, 120, 121, 122, 124, 126, 129, 131, 132, 133, 135, 136, 137,138, 139, 147, 148, 150, 151, 152, 153, 155, 157, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 183, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 201, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, and 236) which allow modification of FcγRbinding properties, effector function, and potentially clinicalproperties of antibodies.

As described in U.S. Ser. No. 11/090,981, filed Mar. 24, 2005, entitled“Immunoglobulin variants outside the Fc region”, and incorporated hereinit its entirety by reference, positional means for optimizing effectorfunction, include but is not limited to, modification of an amino acidat one or more light chain constant region positions (e.g., at positions108, 109, 110, 111, 112, 114, 116, 121, 122, 123, 124, 125, 126, 127,128, 129, 131, 137, 138, 140, 141, 142, 143, 145, 147, 149, 150, 151,152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 176, 180, 181, 182, 183, 184,185, 187, 188, 189, 190, 191, 193, 195, 197, 199, 200, 202, 203, 204,205, 206, 207, 208, 210, 211, 212, 213) which allow modification of FcγRbinding properties, effector function, and potentially clinicalproperties of antibodies.

In particular, substitutional means for altering binding to one or morehuman Fc receptors include, but is not limited to, a substitution of anamino acid at one or more heavy chain constant region positions, e.g.,one or more of the following substitutions: 118K, 118E, 118Y, 119R,119E, 119Y, 120R, 120E, 120I, 121E, 121Y, 121H, 122E, 122R, 124K, 124E,124Y, 126K, 126D, 129L, 129D, 131G, 131T, 132D, 132R, 132L, 133R, 133E,133L, 135I, 135E, 135K, 136E, 136K, 136I, 137E, 138S, 138R, 138D, 139I,139E, 139K, 147A, 147E, 148Y, 148K, 150L, 150K, 150E, 151A, 151D, 152L,152K, 153L, 153D, 155E, 155K, 155I, 157E, 157K, 157Y, 159K, 159D, 159L,160K, 160E, 160Y, 161D, 162D, 162K, 162Y, 163R, 164R, 164E, 164Y, 165D,165R, 165Y, 166D, 167A, 168L, 169E, 171G, 171H, 172K, 172L, 172E, 173T,173D, 174E, 174K, 174Y, 175D, 175L, 176D, 176R, 176L, 177R, 177E, 177Y,178D, 179K, 179Y, 179E, 180K, 180L, 180E, 183T, 187I, 187K, 187E, 188I,189D, 189G, 190I, 190K, 190E, 191D, 191R, 191Y, 192N, 192R, 192L, 193F,193E, 194R, 194D, 195R, 195D, 195Y, 196K, 196D, 196L, 197R, 197E, 197Y,198L, 199T, 199D, 199K, 201E, 201K, 201L, 203D, 203L, 203K, 205D, 205L,206A, 206E, 207K, 207D, 208R, 208E, 208Y, 209E, 209K, 209Y, 210L, 210E,210Y, 211R, 211E, 211Y, 212Q, 212K, 212H, 212L, 212Y, 213N, 213E, 213H,213L, 213Y, 214N, 214E, 214H, 214L, 214Y, 216N, 216K, 216H, 216L, 216Y,217D, 217H, 217A, 217V, 217G, 218D, 218E, 218Q, 218T, 218H, 218L, 218Y,219D, 219E, 219Q, 219K, 219T, 219H, 219L, 219I, 219Y, 205A, 210A, 213A,214A, 218A, 221K, 221Y, 221E, 221N, 221Q, 221R, 221S, 221T, 221H, 221A,221V, 221L, 221I, 221F, 221M, 221W, 221P, 221G, 222E, 222Y, 222D, 222N,222Q, 222R, 222S, 222T, 222H, 222V, 222L, 222I, 222F, 222M, 222W, 222P,222G, 222A, 223D, 223N, 223Q, 223R, 223S, 223H, 223A, 223V, 223L, 223I,223F, 223M, 223Y, 223W, 223P, 223G, 223E, 223K, 224D, 224N, 224Q, 224K,224R, 224S, 224T, 224V, 224L, 224I, 224F, 224M, 224W, 224P, 224G, 224E,224Y, 224A, 225D, 225N, 225Q, 225R, 225S, 225H, 225A, 225V, 225L, 225I,225F, 225M, 225Y, 225P, 225G, 225E, 225K, 225W, 226S, 227E, 227K, 227Y,227G, 227D, 227N, 227Q, 227R, 227S, 227T, 227H, 227A, 227V, 227L, 227I,227F, 227M, 227W, 228K, 228Y, 228G, 228D, 228N, 228Q, 228R, 228T, 228H,228A, 228V, 228L, 228I, 228F, 228M, 228W, 229S, 230A, 230E, 230Y, 230G,230D, 230N, 230Q, 230K, 230R, 230S, 230T, 230H, 230V, 230L, 230I, 230F,230M, 230W, 231K, 231P, 231D, 231N, 231Q, 231R, 231S, 231T, 231H, 231V,231L, 231I, 231F, 231M, 231W, 232E, 232K, 232Y, 232G, 232D, 232N, 232Q,232R, 232S, 232T, 232H, 232A, 232V, 232L, 232I, 232F, 232M, 232W, 233D,233N, 233Q, 233R, 233S, 233T, 233H, 233A, 233V, 233L, 233I, 233F, 233M,233Y, 233W, 233G, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V,234F, 234K, 234R, 234S, 234A, 234M, 234G, 235D, 235S, 235N, 235Q, 235T,235H, 235Y, 235I, 235V, 235F, 235E, 235K, 235R, 235A, 235M, 235W, 235P,235G, 236D, 236E, 236N, 236Q, 236K, 236R, 236S, 236T, 236H, 236A, 236V,236L, 236I, 236F, 236M, 236Y, 236W, and 236P, wherein numbering isaccording to the EU index.

In particular, substitutional means for altering binding to one or morehuman Fc receptors include, but is not limited to, a substitution of anamino acid modification at one or more light chain constant regionpositions, e.g., one or more of the following amino acid substitutionsin the light chain constant region positions: 108D, 108I, 108Q, 109D,109P, 109R, 110E, 110I, 110K, 111E, 111K, 111L, 112E, 112R, 112Y, 114D,114I, 114K, 116T, 121D, 122R, 122S, 122Y, 123L, 123R, 124E, 125E, 125K,126D, 126L, 126Q, 127A, 127D, 127K, 128N, 129E, 129I, 129K, 131T, 137K,137S, 138D, 138K, 138L, 140E, 140H, 140K, 141E, 141K, 142D, 142G, 142L,143A, 143L, 143R, 145D, 145T, 145Y, 147A, 147E, 147K, 149D, 149Y, 150A,151I, 151K, 152L, 152R, 152S, 153D, 153H, 153S, 154E, 154R, 154V, 155E,155I, 155K, 156A, 156D, 156R, 157N, 158D, 158L, 158R, 159E, 159K, 159L,160K, 160V, 161K, 161L, 162T, 163E, 163K, 163T, 164Q, 165K, 165P, 165Y,166E, 166M, 166S, 167K, 167L, 168K, 168Q, 168Y, 169D, 169H, 169S, 170I,170N, 170R, 171A, 171N, 171V, 172E, 172I, 172K, 173K, 173L, 173Q, 174A,176T, 180E, 180K, 180S, 181K, 182E, 182R, 182T, 183D, 183L, 183P, 184E,184K, 184Y, 185I, 185Q, 185R, 187K, 187Y, 188E, 188S, 188Y, 189D, 189K,189Y, 190E, 190L, 190R, 191E, 191R, 191S, 193E, 193K, 193S, 195I, 195K,195Q, 197E, 197K, 197L, 199E, 199K, 199Y, 200S, 202D, 202R, 202Y, 203D,203L, 203R, 204T, 205E, 205K, 206E, 206I, 206K, 207A, 207E, 207L, 208E,208K, 208T, 210A, 210E, 210K, 211A, 211E, 211P, 212E, 212K, 212T, 213L,213R, wherein numbering is according to the EU index.

Additional substitutional means that may also be used in the presentinvention include substitutional means for modulating Fc receptoraffinity, FcγR-mediated effector function, and/or complement mediatedeffector function, e.g., one or more of the following amino acidsubstitutions: 298A, 298T, 326A, 326D, 326E, 326W, 326Y, 333A, 333S,334L, and 334A (U.S. Pat. No. 6,737,056; Shields et al, Journal ofBiological Chemistry, 2001, 276(9):6591-6604; U.S. Pat. No. 6,528,624;Idusogie et al., 2001, J. Immunology 166:2571-2572), 247L, 255L, 270E,392T, 396L, and 421K (U.S. Ser. No. 10/754,922; U.S. Ser. No.10/902,588), and 280H, 280Q, and 280Y (U.S. Ser. No. 10/370,749), eachincorporated herein it its entirety by reference.

In other embodiments, antibodies of the present invention may becombined with means for altering FcRn binding, e.g., antibodies of thepresent invention may be combined with constant heavy chain variants.These include means for modifying FcRn affinity in a pH-specific manner.In particular, substitutional means for increasing Fc binding to FcRninclude, but are not limited to, one or more of the following amino acidsubstitutions: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004,J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal ofImmunology 176:346-356, U.S. Ser. No. 11/102,621, PCT/US2003/033037,PCT/US2004/011213, U.S. Ser. No. 10/822,300, U.S. Ser. No. 10/687,118,PCT/US2004/034440, U.S. Ser. No. 10/966,673, each incorporated herein itits entirety by reference), 256A, 272A, 286A, 305A,0 307A, 311A, 312A,376A, 378Q, 380A, 382A, 434A (Shields et al, Journal of BiologicalChemistry, 2001, 276(9):6591-6604, U.S. Ser. No. 10/982,470, U.S. Pat.No. 6,737,056, U.S. Ser. No. 11/429,793, U.S. Ser. No. 11/429,786,PCT/US2005/029511, U.S. Ser. No. 11/208,422, each incorporated herein itits entirety by reference), 252F, 252T, 252Y, 252W, 254T, 256S, 256R,256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 433I, 433P, 433Q, 434H,434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dall Acquaet al. Journal of Immunology, 2002, 169:5171-5180, U.S. Pat. No.7,083,784, PCT/US97/03321, U.S. Pat. No. 6,821,505, PCT/US01/48432, U.S.Ser. No. 11/397,328, each incorporated herein it its entirety byreference), 257C, 257M, 257L, 257N, 257Y, 279E, 279Q, 279Y, insertion ofSer after 281, 283F, 284E, 306Y, 307V, 308F, 308Y 311V, 385H, 385N,(PCT/US2005/041220, U.S. Ser. No. 11/274,065, U.S. Ser. No. 11/436,266,each incorporated herein it its entirety by reference) 204D, 284E, 285E,286D, and 290E (PCT/US2004/037929 incorporated herein it its entirety byreference).

In some embodiments of the invention, antibodies may comprise means forisotypic modifications, that is modifications in a parent IgG to theamino acid type in an alternate IgG. For example as illustrated in FIG.1, an IgG1/IgG3 hybrid variant may be constructed by a substitutionalmeans for substituting IgG1 positions in the CH2 and/or CH3 region withthe amino acids from IgG3 at positions where the two isotypes differ.Thus a hybrid variant IgG antibody may be constructed that comprises oneor more substitutional means, e.g., 274Q, 276K, 300F, 339T, 356E, 358M,384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments of theinvention, an IgG1/IgG2 hybrid variant may be constructed by asubstitutional means for substituting IgG2 positions in the CH2 and/orCH3 region with amino acids from IgG1 at positions where the twoisotypes differ. Thus a hybrid variant IgG antibody may be constructedthat comprises one or more substitutional means, e.g., one or more ofthe following amino acid substations: 233E, 234L, 235L, −236G (referringto an insertion of a glycine at position 236), and 327A.

Glycoform Modifications

Many polypeptides, including antibodies, are subjected to a variety ofpost-translational modifications involving carbohydrate moieties, suchas glycosylation with oligosaccharides. There are several factors thatcan influence glycosylation. The species, tissue and cell type have allbeen shown to be important in the way that glycosylation occurs. Inaddition, the extracellular environment, through altered cultureconditions such as serum concentration, may have a direct effect onglycosylation. (Lifely et al., 1995, Glycobiology 5(8): 813-822),incorporated herein it its entirety by reference.

All antibodies contain carbohydrate at conserved positions in theconstant regions of the heavy chain. Each antibody isotype has adistinct variety of N-linked carbohydrate structures. Aside from thecarbohydrate attached to the heavy chain, up to 30% of human IgGs have aglycosylated Fab region. IgG has a single N-linked biantennarycarbohydrate at Asn297 of the CH2 domain. For IgG from either serum orproduced ex vivo in hybridomas or engineered cells, the IgG areheterogeneous with respect to the Asn297 linked carbohydrate (Jefferiset al., 1998, Immunol. Rev. 163:59-76; Wright et al., 1997, TrendsBiotech 15:26-32, each incorporated herein it its entirety byreference). For human IgG, the core oligosaccharide normally consists ofGlcNAc₂Man₃GlcNAc, with differing numbers of outer residues.

The carbohydrate moieties of the present invention will be describedwith reference to commonly used nomenclature for the description ofoligosaccharides. A review of carbohydrate chemistry which uses thisnomenclature is found in Hubbard et al. 1981, Ann. Rev. Biochem.50:555-583, incorporated herein it its entirety by reference. Thisnomenclature includes, for instance, Man, which represents mannose;GlcNAc, which represents 2-N-acetylglucosamine; Gal which representsgalactose; Fuc for fucose; and Glc, which represents glucose. Sialicacids are described by the shorthand notation NeuNAc, for5-N-acetylneuraminic acid, and NeuNGc for 5-glycolylneuraminic.

The term “glycosylation” is the attachment of oligosaccharides(carbohydrates containing two or more simple sugars linked together e.g.from two to about twelve simple sugars linked together) to aglycoprotein. The oligosaccharide side chains are typically linked tothe backbone of the glycoprotein through either N- or O-linkages. Theoligosaccharides of the present invention occur generally are attachedto a CH2 domain of an Fc region as N-linked oligosaccharides. “N-linkedglycosylation” refers to the attachment of the carbohydrate moiety to anasparagine residue in a glycoprotein chain. The skilled artisan willrecognize that, for example, each of murine IgG1, IgG2a, IgG2b and IgG3as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2 domains have asingle site for N-linked glycosylation at amino acid residue 297 (Kabatet al. Sequences of Proteins of Immunological Interest, 1991,incorporated herein it its entirety by reference).

For the purposes herein, a “mature core carbohydrate structure” refersto a processed core carbohydrate structure attached to an Fc regionwhich generally consists of the following carbohydrate structureGlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)₂ typical of biantennaryoligosaccharides. The mature core carbohydrate structure is attached tothe Fc region of the glycoprotein, generally via N-linkage to Asn297 ofa CH2 domain of the Fc region. A “bisecting GlcNAc” is a GlcNAc residueattached to the β1,4 mannose of the mature core carbohydrate structure.The bisecting GlcNAc can be enzymatically attached to the mature corecarbohydrate structure by a β(1,4)-N-acetylglucosaminyltransferase IIIenzyme (GnTIII). CHO cells do not normally express GnTIII (Stanley etal., 1984, J. Biol. Chem. 261:13370-13378), but may be engineered to doso (Umana et al., 1999, Nature Biotech. 17:176-180).

The present invention contemplates antibodies that comprise modifiedglycoforms or engineered glycoforms. By “modified glycoform” or“engineered glycoform” as used herein is meant a carbohydratecomposition that is covalently attached to a protein, for example anantibody, wherein the carbohydrate composition differs chemically fromthat of a parent protein. Engineered glycoforms may be useful for avariety of purposes, including but not limited to enhancing or reducingFcγR-mediated effector function. In one embodiment, the antibodies ofthe present invention are modified to control the level of fucosylatedand/or bisecting oligosaccharides that are covalently attached to the Fcregion.

Historically, antibodies produced in Chinese Hamster Ovary Cells (CHO),one of the most commonly used industrial hosts, contain about 2 to 6% inthe population that are nonfucosylated. YB2/0 (rat myeloma) and Lec13cell line (a lectin mutant of CHO line which has a deficient GDP-mannose4,6 dehydratase leading to the deficiency of GDP-fucose or GDP-sugarintermediates that are the substrate of α1,6-fucosyltransferase (Ripkaet al., 1986), however, can produce antibodies with 78% to 98%nonfucosylated species. Unfortunately, the yield of antibody from thesecells is extremely poor and therefore these cell lines are not useful tomake therapeutic antibody products commercially. The FUT8 gene encodesthe α1,6-fucosyltransferase enzyme that catalyzes the transfer of afucosyl residue from GDP-fucose to position 6 of Asn-linked (N-linked)GlcNac of an N-glycan (Yanagidani et al., 1997, J Biochem 121:626-632).It is known that the α1,6-fucosyltransferase is the only enzymeresponsible for adding fucose to the N-linked biantennary carbohydrateat Asn297 in the CH2 domain of the IgG antibody.

A variety of methods are well known in the art for generating modifiedglycoforms (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies etal., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473);(U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCTWO 02/30954A1); Yamane-Ohnuki et al., 2004, Biotechnology andBioengineering 87(5):614-621; (Potelligent™ technology [Biowa, Inc.,Princeton, N.J.]; GlycoMAb™ glycosylation engineering technology[GLYCART biotechnology AG, Zurich, Switzerland]; all of which areexpressly incorporated by reference). These techniques control the levelof fucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells), by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed.

Other methods for modifying glycoforms of the antibodies of theinvention include using glycoengineered strains of yeast (Li et al.,2006, Nature Biotechnology 24(2):210-215), moss (Nechansky et al., 2007,Mol Immunjol 44(7):1826-8), and plants (Cox et al., 2006, Nat Biotechnol24(12):1591-7). Methods for modifying glycoforms include but are notlimited to using a glycoengineered strain of yeast Pichia pastoris (Liet al., 2006, Nature Biotechnology 24(2):210-215), a glycoengineeredstrain of the moss Physcomitrella patens wherein the enzymesβ1,2-xylosyltransferase and/or α1,3-fucosyltransferase are knocked outin (Nechansky et al., 2007, Mol. Immunol. 44(7):1826-8), and the use ofRNA interference to inhibit endogenous alpha-1,3-fucosyltransferaseand/or beta-1,2-xylosyltransferase in the aquatic plant Lemna minor (Coxet al., 2006, Nat. Biotechnol. 24(12):1591-7).

Engineered glycoform typically refers to the different carbohydrate oroligosaccharide; thus for example an antibody may comprise an engineeredglycoform. Alternatively, engineered glycoform may refer to the antibodythat comprises the different carbohydrate or oligosaccharide. For thepurposes of modified glycoforms described herein, a “parent antibody” isa glycosylated antibody having the same amino acid sequence and maturecore carbohydrate structure as an engineered glycoform of the presentinvention, except that fucose is attached to the mature corecarbohydrate structure of the parent antibody. For instance, in acomposition comprising the parent glycoprotein about 50-100% or about70-100% of the parent glycoprotein comprises a mature core carbohydratestructure having fucose attached thereto.

The present invention provides a composition comprising a glycosylatedantibody having an Fc region, wherein about 51-100% of the glycosylatedantibody in the composition comprises a mature core carbohydratestructure which lacks fucose, attached to the Fc region of the antibody.In one embodiment, about 80-100% of the antibody in the compositioncomprises a mature core carbohydrate structure which lacks fucose. Inanother embodiment about 90-99% of the antibody in the composition lacksfucose attached to the mature core carbohydrate structure. In anotherembodiment, the antibody in the composition both comprises a mature corecarbohydrate structure that lacks fucose and additionally comprises atleast one amino acid modification in the Fc region. In anotherembodiment, the combination of engineered glycoform and amino acidmodification provides optimal Fc receptor binding properties to theanti-CD40 antibody.

Optimized Properties of Antibodies

The present invention provides variant antibodies that are optimized fora number of therapeutically relevant properties. A variant antibodycomprises one or more amino acid modifications or glycoformmodifications relative to a parent antibody, wherein the amino acidmodification(s) provide one or more optimized properties. Thus theantibodies of the present invention are variant antibodies. An antibodyof the present invention differs in amino acid sequence from its parentantibody by virtue of at least one amino acid or glycoform modification.Thus variant antibodies of the present invention have at least one aminoacid or glycoform modification compared to the parent. Alternatively,the variant antibodies of the present invention may have more than oneamino acid modification as compared to the parent, for example fromabout one to fifty amino acid modifications, e.g., from about one to tenamino acid modifications, from about one to about five amino acidmodifications, etc., compared to the parent. Thus the sequences of thevariant antibodies and those of the parent antibodies are substantiallyhomologous. For example, the variant antibody sequences herein willpossess about 80% homology with the parent antibody sequence, e.g., atleast about 90% homology, e at least about 95% homology, etc.

In another embodiment, the antibodies of the present invention compriseamino acid modifications that provide optimized effector functionproperties relative to the parent. Substitutions and optimized effectorfunction properties are described in U.S. Ser. No. 10/672,280, PCTUS03/30249, and U.S. Ser. No. 10/822,231, and U.S. Ser. No. 60/627,774,filed Nov. 12/2004 and entitled “Optimized Fc Variants”. Properties thatmay be optimized include but are not limited to enhanced or reducedaffinity for an FcγR. In one embodiment, the antibodies of the presentinvention are optimized to possess enhanced affinity for a humanactivating FcγR, e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb.In one embodiment, an antibody of the invention are optimized to possessenhanced affinity for a human FcγRIIIa. In an alternate embodiment, theantibodies are optimized to possess reduced affinity for the humaninhibitory receptor FcγRIIb. These embodiments are anticipated toprovide antibodies with enhanced therapeutic properties in humans, forexample enhanced effector function and greater anti-cancer potency. Inan alternate embodiment, the antibodies of the present invention areoptimized to have reduced or ablated affinity for a human FcγR,including but not limited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa,and FcγRIIIb. These embodiments are anticipated to provide antibodieswith enhanced therapeutic properties in humans, for example reducedeffector function and reduced toxicity. In other embodiments, antibodiesof the present invention provide enhanced affinity for one or moreFcγRs, yet reduced affinity for one or more other FcγRs. For example, anantibody of the present invention may have enhanced binding to FcγRIIIa,yet reduced binding to FcγRIIb. Alternately, an antibody of the presentinvention may have enhanced binding to FcγRIIa and FcγRI, yet reducedbinding to FcγRIIb. In yet another embodiment, an antibody of thepresent invention may have enhanced affinity for FcγRIIb, yet reducedaffinity to one or more activating FcγRs.

The modifications of the invention may enhance binding affinity for oneor more FcγRs. By “greater affinity” or “improved affinity” or “enhancedaffinity” or “better affinity” than a parent immunoglobulin, as usedherein is meant that an Fc variant binds to an Fc receptor with asignificantly higher equilibrium constant of association (K_(a)) orlower equilibrium constant of dissociation (K_(d)) than the parentpolypeptide when the amounts of variant and parent polypeptide in thebinding assay are essentially the same. For example, the Fc variant withimproved FcγR binding affinity may display from about 5 fold to about1000 fold, e.g. from about 10 fold to about 500 fold improvement in Fcreceptor binding affinity compared to the parent polypeptide, where Fcreceptor binding affinity is determined, for example, as disclosed inthe Examples herein. Accordingly, by “reduced affinity” as compared to aparent Fc polypeptide as used herein is meant that an Fc variant bindsan Fc receptor with significantly lower K_(a) or higher K_(d) than theparent polypeptide.

Embodiments comprise optimization of Fc binding to a human FcγR, howeverin alternate embodiments the antibodies of the present invention possessenhanced or reduced affinity for FcγRs from nonhuman organisms,including but not limited to rodents and non-human primates. Antibodiesthat are optimized for binding to a nonhuman FcγR may find use inexperimentation. For example, mouse models are available for a varietyof diseases that enable testing of properties such as efficacy,toxicity, and pharmacokinetics for a given drug candidate. As is knownin the art, cancer cells can be grafted or injected into mice to mimic ahuman cancer, a process referred to as xenografting. Testing ofantibodies that comprise antibodies that are optimized for one or moremouse FcγRs, may provide valuable information with regard to theefficacy of the protein, its mechanism of action, and the like. Theantibodies of the present invention may also be optimized for enhancedfunctionality and/or solution properties in aglycosylated form. In oneembodiment, the aglycosylated antibodies of the present invention bindan Fc ligand with greater affinity than the aglycosylated form of theparent antibody. The Fc ligands include but are not limited to FcγRs,C1q, FcRn, and proteins A and G, and may be from any source includingbut not limited to human, mouse, rat, rabbit, or monkey. In an alternateembodiment, the antibodies are optimized to be more stable and/or moresoluble than the aglycosylated form of the parent antibody.

Antibodies of the invention may comprise modifications that modulateinteraction with Fc ligands other than FcγRs, including but not limitedto complement proteins, FcRn, and Fc receptor homologs (FcRHs). FcRHsinclude but are not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, andFcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136, incorporatedherein it its entirety by reference).

In one embodiment, the Fc ligand specificity of the antibody of thepresent invention will determine its therapeutic utility. The utility ofa given antibody for therapeutic purposes will depend on the epitope orform of the target antigen and the disease or indication being treated.For some targets and indications, enhanced FcγR-mediated effectorfunctions may be desirable. This may be particularly favorable foranti-cancer antibodies. Thus antibodies may be used that compriseantibodies that provide enhanced affinity for activating FcγRs and/orreduced affinity for inhibitory FcγRs. For some targets and indications,it may be further beneficial to utilize antibodies that providedifferential selectivity for different activating FcγRs; for example, insome cases enhanced binding to FcγRIIa and FcγRIIIa may be desired, butnot FcγRI, whereas in other cases, enhanced binding only to FcγRIIa maybe desired. For certain targets and indications, it may be desirable toutilize antibodies that enhance both FcγR-mediated andcomplement-mediated effector functions, whereas for other cases it maybe advantageous to utilize antibodies that enhance either FcγR-mediatedor complement-mediated effector functions. For some targets or cancerindications, it may be advantageous to reduce or ablate one or moreeffector functions, for example by knocking out binding to C1q, one ormore FcγR's, FcRn, or one or more other Fc ligands. For other targetsand indications, it may be desirable to utilize antibodies that provideenhanced binding to the inhibitory FcγRIIb, WT level, reduced, orablated binding to activating FcγRs. This may be particularly useful,for example, when the goal of an antibody is to inhibit inflammation orauto-immune disease, or modulate the immune system in some way.

Clearly an important parameter that determines the most beneficialselectivity of a given antibody to treat a given disease is the contextof the antibody, that is what type of antibody is being used. Thus theFc ligand selectivity or specificity of a given antibody will providedifferent properties depending on whether it composes an antibody or anantibodies with a coupled fusion or conjugate partner. For example,toxin, radionucleotide, or other conjugates may be less toxic to normalcells if the antibody that comprises them has reduced or ablated bindingto one or more Fc ligands. As another example, in order to inhibitinflammation or auto-immune disease, it may be desirable to utilize anantibody with enhanced affinity for activating FcγRs, such as to bindthese FcγRs and prevent their activation. Conversely, an antibody thatcomprises two or more Fc regions with enhanced FcγRIIb affinity mayco-engage this receptor on the surface of immune cells, therebyinhibiting proliferation of these cells. Whereas in some cases anantibodies may engage its target antigen on one cell type yet engageFcγRs on separate cells from the target antigen, in other cases it maybe advantageous to engage FcγRs on the surface of the same cells as thetarget antigen. For example, if an antibody targets an antigen on a cellthat also expresses one or more FcγRs, it may be beneficial to utilizean antibody that enhances or reduces binding to the FcγRs on the surfaceof that cell. This may be the case, for example when the antibody isbeing used as an anti-cancer agent, and co-engagement of target antigenand FcγR on the surface of the same cell promote signaling events withinthe cell that result in growth inhibition, apoptosis, or otheranti-proliferative effect. Alternatively, antigen and FcγR co-engagementon the same cell may be advantageous when the antibody is being used tomodulate the immune system in some way, wherein co-engagement of targetantigen and FcγR provides some proliferative or anti-proliferativeeffect. Likewise, antibodies that comprise two or more Fc regions maybenefit from antibodies that modulate FcγR selectivity or specificity toco-engage FcγRs on the surface of the same cell.

The Fc ligand specificity of the antibodies of the present invention canbe modulated to create different effector function profiles that may besuited for particular antigen epitopes, indications or patientpopulations. FIG. 3 describes several embodiments of receptor bindingprofiles that include improvements to, reductions to or no effect to thebinding to various receptors, where such changes may be beneficial incertain contexts. The receptor binding profiles in the figure could bevaried by degree of increase or decrease to the specified receptors.Additionally, the binding changes specified could be in the context ofadditional binding changes to other receptors such as C1q or FcRn, forexample by combining with ablation of binding to C1q to shut offcomplement activation, or by combining with enhanced binding to C1q toincrease complement activation. Other embodiments with other receptorbinding profiles are possible, the listed receptor binding profiles areexemplary.

The presence of different polymorphic forms of FcγRs provides yetanother parameter that impacts the therapeutic utility of the antibodiesof the present invention. Whereas the specificity and selectivity of agiven antibody for the different classes of FcγRs significantly affectsthe capacity of an antibody to target a given antigen for treatment of agiven disease, the specificity or selectivity of an antibody fordifferent polymorphic forms of these receptors may in part determinewhich research or pre-clinical experiments may be appropriate fortesting, and ultimately which patient populations may or may not respondto treatment. Thus the specificity or selectivity of antibodies of thepresent invention to Fc ligand polymorphisms, including but not limitedto FcγR, C1q, FcRn, and FcRH polymorphisms, may be used to guide theselection of valid research and pre-clinical experiments, clinical trialdesign, patient selection, dosing dependence, and/or other aspectsconcerning clinical trials.

Other Modifications

Antibodies of the present invention may comprise one or moremodifications that provide optimized properties that are notspecifically related to effector function per se. The modifications maybe amino acid modifications, or may be modifications that are madeenzymatically or chemically. Such modification(s) likely provide someimprovement in the antibody, for example an enhancement in itsstability, solubility, function, or clinical use. The present inventioncontemplates a variety of improvements that made be made by coupling theantibodies of the present invention with additional modifications.

In one embodiment, the variable region of an antibody of the presentinvention may be affinity matured, that is to say that amino acidmodifications have been made in the VH and/or VL domains of the antibodyto enhance binding of the antibody to its target antigen. Such types ofmodifications may improve the association and/or the dissociationkinetics for binding to the target antigen. Other modifications includethose that improve selectivity for target antigen vs. alternativetargets. These include modifications that improve selectivity forantigen expressed on target vs. non-target cells. Other improvements tothe target recognition properties may be provided by additionalmodifications. Such properties may include, but are not limited to,specific kinetic properties (i.e. association and dissociationkinetics), selectivity for the particular target versus alternativetargets, and selectivity for a specific form of target versusalternative forms. Examples include full-length versus splice variants,cell-surface vs. soluble forms, selectivity for various polymorphicvariants, or selectivity for specific conformational forms of the targetantigen.

Antibodies of the invention may comprise one or more modifications thatprovide reduced or enhanced internalization of an antibody. In oneembodiment, antibodies of the present invention can be utilized orcombined with additional modifications in order to reduce the cellularinternalization of an antibody that occurs via interaction with one ormore Fc ligands. This property might be expected to enhance effectorfunction, and potentially reduce immunogenicity of the antibodies of theinvention. Alternatively, antibodies of the present antibodies of thepresent invention can be utilized directly or combined with additionalmodifications in order to enhance the cellular internalization of anantibody that occurs via interaction with one or more Fc ligands. Forexample, in one embodiment, an antibody is used that provides enhancedbinding to FcγRI, which is expressed on dendritic cells and active earlyin immune response. This strategy could be further enhanced bycombination with additional modifications, either within the antibody orin an attached fusion or conjugate partner, that promote recognition andpresentation of Fc peptide fragments by MHC molecules. These strategiesare expected to enhance target antigen processing and thereby improveantigenicity of the target antigen (Bonnerot and Amigorena, 1999,Immunol Rev. 172:279-84, incorporated herein it its entirety byreference), promoting an adaptive immune response and greater targetcell killing by the human immune system. These strategies may beparticularly advantageous when the targeted antigen is shed from thecellular surface. An additional application of these concepts ariseswith idiotype vaccine immunotherapies, in which clone-specificantibodies produced by a patient's lymphoma cells are used to vaccinatethe patient.

In one embodiment, modifications are made to improve biophysicalproperties of the antibodies of the present invention, including but notlimited to stability, solubility, and oligomeric state. Modificationscan include, for example, substitutions that provide more favorableintramolecular interactions in the antibody such as to provide greaterstability, or substitution of exposed nonpolar amino acids with polaramino acids for higher solubility. A number of optimization goals andmethods are described in U.S. Ser. No. 10/379,392, incorporated hereinit its entirety by reference, that may find use for engineeringadditional modifications to further optimize the antibodies of thepresent invention. The antibodies of the present invention can also becombined with additional modifications that reduce oligomeric state orsize, such that tumor penetration is enhanced, or in vivo clearancerates are increased as desired.

Other modifications to the antibodies of the present invention includethose that enable the specific formation or homodimeric orhomomultimeric molecules. Such modifications include but are not limitedto engineered disulfides, as well as chemical modifications oraggregation methods which may provide a mechanism for generatingcovalent homodimeric or homomultimers. For example, methods ofengineering and compositions of such molecules are described in Kan etal., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002,Recent Results Cancer Res. 159: 104-12; U.S. Pat. No. 5,681,566; Caronet al., 1992, J. Exp. Med. 176:1191-1195, and Shopes, 1992, J. Immunol.148(9):2918-22, each incorporated herein it its entirety by reference.Additional modifications to the variants of the present inventioninclude those that enable the specific formation or heterodimeric,heteromultimeric, bifunctional, and/or multifunctional molecules. Suchmodifications include, but are not limited to, one or more amino acidsubstitutions in the CH3 domain, in which the substitutions reducehomodimer formation and increase heterodimer formation. For example,methods of engineering and compositions of such molecules are describedin Atwell et al., 1997, J. Mol. Biol. 270(1):26-35, and Carter et al.,2001, J. Immunol. Methods 248:7-15, each incorporated herein it itsentirety by reference. Additional modifications include modifications inthe hinge and CH3 domains, in which the modifications reduce thepropensity to form dimers.

In further embodiments, the antibodies of the present invention comprisemodifications that remove proteolytic degradation sites. These mayinclude, for example, protease sites that reduce production yields, aswell as protease sites that degrade the administered protein in vivo. Inone embodiment, additional modifications are made to remove covalentdegradation sites such as deamidation (i.e. deamidation of glutaminyland asparaginyl residues to the corresponding glutamyl and aspartylresidues), oxidation, and proteolytic degradation sites. Deamidationsites that are particularly useful to remove are those that have enhancepropensity for deamidation, including, but not limited to asparaginyland glutamyl residues followed by glycines (NG and QG motifs,respectively). In such cases, substitution of either residue cansignificantly reduce the tendency for deamidation. Common oxidationsites include methionine and cysteine residues. Other covalentmodifications, that can either be introduced or removed, includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the “-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983), incorporated herein it its entirety by reference),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group. Additional modifications also may include but are notlimited to posttranslational modifications such as N-linked or O-linkedglycosylation and phosphorylation.

Modifications may include those that improve expression and/orpurification yields from hosts or host cells commonly used forproduction of biologics. These include, but are not limited to variousmammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines,and plants. Additional modifications include modifications that removeor reduce the ability of heavy chains to form inter-chain disulfidelinkages. Additional modifications include modifications that remove orreduce the ability of heavy chains to form intra-chain disulfidelinkages.

The antibodies of the present invention may comprise modifications thatinclude the use of unnatural amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci.U.S.A. 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin etal., 2003, Science 301(5635):964-7, each incorporated herein it itsentirety by reference. In some embodiments, these modifications enablemanipulation of various functional, biophysical, immunological, ormanufacturing properties discussed above. In additional embodiments,these modifications enable additional chemical modification for otherpurposes. Other modifications are contemplated herein. For example, theantibody may be linked to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes,or copolymers of polyethylene glycol and polypropylene glycol.Additional amino acid modifications may be made to enable specific ornon-specific chemical or posttranslational modification of theantibodies. Such modifications, include, but are not limited toPEGylation and glycosylation. Specific substitutions that can beutilized to enable PEGylation include, but are not limited to,introduction of novel cysteine residues or unnatural amino acids suchthat efficient and specific coupling chemistries can be used to attach aPEG or otherwise polymeric moiety. Introduction of specificglycosylation sites can be achieved by introducing novel N-X-T/Ssequences into the antibodies of the present invention.

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

In some embodiments, the covalent modification of the antibodies of theinvention comprises the addition of one or more labels. The term“labeling group” is any detectable label. In some embodiments, thelabeling group is coupled to the antibody via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used in performing thepresent invention. In general, labels fall into a variety of classes,depending on the assay in which they are to be detected: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic labels(e.g., magnetic particles); c) redox active moieties; d) optical dyes;enzymatic groups (e.g. horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase); e) biotinylated groups; and f)predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags, etc.). In someembodiments, the labeling group is coupled to the antibody via spacerarms of various lengths to reduce potential steric hindrance. Variousmethods for labeling proteins are known in the art and may be used inperforming the present invention. Specific labels include optical dyes,including, but not limited to, chromophores, phosphors and fluorophores,with the latter being specific in many instances. Fluorophores can beeither “small molecule” fluores, or proteinaceous fluores. By“fluorescent label” is meant any molecule that may be detected via itsinherent fluorescent properties.

Antibody Conjugates and Fusions

In one embodiment, the antibodies of the invention are antibody “fusionproteins”, sometimes referred to herein as “antibody conjugates”. Thefusion partner or conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antibody and on the conjugate partner. Conjugate andfusion partners may be any molecule, including small molecule chemicalcompounds and polypeptides. For example, a variety of antibodyconjugates and methods are described in Trail et al., 1999, Curr. Opin.Immunol. 11:584-588, incorporated herein it its entirety by reference.Possible conjugate partners include but are not limited to cytokines,cytotoxic agents, toxins, radioisotopes, chemotherapeutic agent,anti-angiogenic agents, a tyrosine kinase inhibitors, and othertherapeutically active agents. In some embodiments, conjugate partnersmay be thought of more as payloads, that is to say that the goal of aconjugate is targeted delivery of the conjugate partner to a targetedcell, for example a cancer cell or immune cell, by the antibody. Thus,for example, the conjugation of a toxin to an antibody targets thedelivery of the toxin to cells expressing the target antigen. As will beappreciated by one skilled in the art, in reality the concepts anddefinitions of fusion and conjugate are overlapping. The designation ofan antibody as a fusion or conjugate is not meant to constrain it to anyparticular embodiment of the present invention. Rather, these terms areused loosely to convey the broad concept that any antibody of thepresent invention may be linked genetically, chemically, or otherwise,to one or more polypeptides or molecules to provide some desirableproperty.

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody. Additionalembodiments utilize calicheamicin, auristatins, geldanamycin,maytansine, and duocarmycins and analogs; for the latter, see U.S.2003/0050331, incorporated herein it its entirety by reference.

In one embodiment, the antibodies of the present invention are fused orconjugated to a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. For example, as described inPenichet et al., 2001, J. Immunol. Methods 248:91-101, incorporatedherein it its entirety by reference, cytokines may be fused to antibodyto provide an array of desirable properties. Examples of such cytokinesare lymphokines, monokines, and traditional polypeptide hormones.Included among the cytokines are growth hormone such as human growthhormone, N-methionyl human growth hormone, and bovine growth hormone;parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-beta; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; C5a; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines.

In an alternate embodiment, the antibodies of the present invention arefused, conjugated, or operably linked to a toxin, including but notlimited to small molecule toxins and enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof. For example, a variety of immunotoxins and immunotoxinmethods are described in Thrush et al., 1996, Ann. Rev. Immunol.14:49-71, incorporated herein it its entirety by reference. Smallmolecule toxins include but are not limited to calicheamicin, maytansine(U.S. Pat. No. 5,208,020, incorporated herein it its entirety byreference), trichothene, and CC1065. In one embodiment of the invention,the antibody is conjugated to one or more maytansine molecules (e.g.about 1 to about 10 maytansine molecules per antibody molecule).Maytansine may, for example, be converted to May-SS-Me which may bereduced to May-SH3 and reacted with modified antibody (Chari et al.,1992, Cancer Research 52: 127-131, incorporated herein it its entiretyby reference) to generate a maytansinoid-antibody conjugate. Anotherconjugate of interest comprises an antibody conjugated to one or morecalicheamicin molecules. The calicheamicin family of antibiotics arecapable of producing double-stranded DNA breaks at sub-picomolarconcentrations. Structural analogues of calicheamicin that may be usedinclude but are not limited to γ₁ ¹, α₂ ¹, α₃, N-acetyl-yl¹, PSAG, andΘ¹ ₁, (Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al.,1998, Cancer Research 58:2925-2928) (U.S. Pat. No. 5,714,586; U.S. Pat.No. 5,712,374; U.S. Pat. No. 5,264,586; U.S. Pat. No. 5,773,001, eachincorporated herein it its entirety by reference). Dolastatin 10 analogssuch as auristatin E (AE) and monomethylauristatin E (MMAE) may find useas conjugates for the antibodies of the present invention (Doronina etal., 2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood102(4):1458-65, each incorporated herein it its entirety by reference).Useful enzymatically active toxins include but are not limited todiphtheria A chain, nonbinding active fragments of diphtheria toxin,exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, PCT WO 93/21232,incorporated herein it its entirety by reference. The present inventionfurther contemplates a conjugate between an antibody of the presentinvention and a compound with nucleolytic activity, for example aribonuclease or DNA endonuclease such as a deoxyribonuclease (Dnase).

In an alternate embodiment, an antibody of the present invention may befused, conjugated, or operably linked to a radioisotope to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugate antibodies. Examples include, but are notlimited to, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, andradioactive isotopes of Lu.

In yet another embodiment, an antibody of the present invention may beconjugated to a “receptor” (such as streptavidin) for utilization intumor pretargeting wherein the antibody-receptor conjugate isadministered to the patient, followed by removal of unbound conjugatefrom the circulation using a clearing agent and then administration of a“ligand” (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. aradionucleotide). In an alternate embodiment, the antibody is conjugatedor operably linked to an enzyme in order to employ Antibody DependentEnzyme Mediated Prodrug Therapy (ADEPT). ADEPT may be used byconjugating or operably linking the antibody to a prodrug-activatingenzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent,see PCT WO 81/01145, incorporated herein it its entirety by reference)to an active anti-cancer drug. See, for example, PCT WO 88/07378 andU.S. Pat. No. 4,975,278, each incorporated herein it its entirety byreference. The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way so asto covert it into its more active, cytotoxic form. Enzymes that areuseful in the method of this invention include but are not limited toalkaline phosphatase useful for converting phosphate-containing prodrugsinto free drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuramimidase useful for converting glycosylatedprodrugs into free drugs; beta-lactamase useful for converting drugsderivatized with α-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, for example, Massey, 1987, Nature 328: 457-458, incorporatedherein it its entirety by reference). Antibody-abzyme conjugates can beprepared for delivery of the abzyme to a tumor cell population. Avariety of additional conjugates are contemplated for the antibodies ofthe present invention. A variety of chemotherapeutic agents,anti-angiogenic agents, tyrosine kinase inhibitors, and othertherapeutic agents are described below, which may find use as antibodyconjugates.

Also contemplated as fusion and conjugate partners are Fc polypeptides.Thus an antibody may be a multimeric Fc polypeptide, comprising two ormore Fc regions. The advantage of such a molecule is that it providesmultiple binding sites for Fc receptors with a single protein molecule.In one embodiment, Fc regions may be linked using a chemical engineeringapproach. For example, Fab's and Fc's may be linked by thioether bondsoriginating at cysteine residues in the hinges, generating moleculessuch as FabFc₂. Fc regions may be linked using disulfide engineeringand/or chemical cross-linking. In one embodiment, Fc regions may belinked genetically. In one embodiment, Fc regions in an antibody arelinked genetically to generated tandemly linked Fc regions as describedin U.S. Ser. No. 11/022,289, filed Dec. 21, 2004, entitled “Fcpolypeptides with novel Fc ligand binding sites,” incorporated herein itits entirety by reference. Tandemly linked Fc polypeptides may comprisetwo or more Fc regions, e.g., one to three, two, etc., Fc regions. Itmay be advantageous to explore a number of engineering constructs inorder to obtain homo- or hetero-tandemly linked antibodies with the mostfavorable structural and functional properties. Tandemly linkedantibodies may be homo-tandemly linked antibodies, that is an antibodyof one isotype is fused genetically to another antibody of the sameisotype. It is anticipated that because there are multiple FcγR, C1q,and/or FcRn binding sites on tandemly linked Fc polypeptides, effectorfunctions and/or pharmacokinetics may be enhanced. In an alternateembodiment, antibodies from different isotypes may be tandemly linked,referred to as hetero-tandemly linked antibodies. For example, becauseof the capacity to target FcγR and FcαRI receptors, an antibody thatbinds both FcγRs and FcαRI may provide a significant clinicalimprovement.

In addition to antibodies, an antibody-like protein that is finding anexpanding role in research and therapy is the Fc fusion (Chamow et al.,1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr OpinImmunol 9:195-200, each incorporated herein it its entirety byreference). “Fc fusion” is herein meant to be synonymous with the terms“immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin”(sometimes with dashes) as used in the prior art (Chamow et al., 1996,Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200). An Fc fusion is a protein wherein one or more polypeptidesis operably linked to Fc. An Fc fusion combines the Fc region of anantibody, and thus its favorable effector functions andpharmacokinetics, with the target-binding region of a receptor, ligand,or some other protein or protein domain. The role of the latter is tomediate target recognition, and thus it is functionally analogous to theantibody variable region. Because of the structural and functionaloverlap of Fc fusions with antibodies, the discussion on antibodies inthe present invention extends also to Fc.

Virtually any protein or small molecule may be linked to Fc to generatean Fc fusion. Protein fusion partners may include, but are not limitedto, the variable region of any antibody, the target-binding region of areceptor, an adhesion molecule, a ligand, an enzyme, a cytokine, achemokine, or some other protein or protein domain. Small moleculefusion partners may include any therapeutic agent that directs the Fcfusion to a therapeutic target. Such targets may be any molecule, e.g.,an extracellular receptor, that is implicated in disease.

Fusion and conjugate partners may be linked to any region of an antibodyof the present invention, including at the N- or C-termini, or at someresidue in-between the termini. In one embodiment, a fusion or conjugatepartner is linked at the N- or C-terminus of the antibody, e.g., theN-terminus. A variety of linkers may find use in the present inventionto covalently link antibodies to a fusion or conjugate partner. By“linker”, “linker sequence”, “spacer”, “tethering sequence” orgrammatical equivalents thereof, herein is meant a molecule or group ofmolecules (such as a monomer or polymer) that connects two molecules andoften serves to place the two molecules in a desirable configuration.Linkers are known in the art; for example, homo- or hetero-bifunctionallinkers as are well known (see, 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinit its entirety by reference). A number of strategies may be used tocovalently link molecules together. These include, but are not limitedto polypeptide linkages between N- and C-termini of proteins or proteindomains, linkage via disulfide bonds, and linkage via chemicalcross-linking reagents. In one aspect of this embodiment, the linker isa peptide bond, generated by recombinant techniques or peptidesynthesis. The linker may contain amino acid residues that provideflexibility. Thus, the linker peptide may predominantly include thefollowing amino acid residues: Gly, Ser, Ala, or Thr. The linker peptideshould have a length that is adequate to link two molecules in such away that they assume the correct conformation relative to one another sothat they retain the desired activity. Suitable lengths for this purposeinclude at least one and not more than 50 amino acid residues. In oneembodiment, the linker is from about 1 to 30 amino acids in length, withlinkers of 1 to 20 amino acids in length being desirable. Useful linkersinclude glycine-serine polymers (including, for example, (GS)n, (GSGGS)n(GGGGS)n and (GGGS)n, where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers, as will be appreciated by those in the art. Alternatively, avariety of nonproteinaceous polymers, including but not limited topolyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol, may find useas linkers, that is may find use to link the antibodies of the presentinvention to a fusion or conjugate partner, or to link the antibodies ofthe present invention to a conjugate.

Production of Antibodies

The present invention provides methods for producing and experimentallytesting antibodies. The described methods are not meant to constrain thepresent invention to any particular application or theory of operation.Rather, the provided methods are meant to illustrate generally that oneor more antibodies may be produced and experimentally tested to obtainvariant antibodies. General methods for antibody molecular biology,expression, purification, and screening are described in AntibodyEngineering, edited by Duebel & Kontermann, Springer-Verlag, Heidelberg,2001; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689;Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; Antibodies: ALaboratory Manual by Harlow & Lane, New York: Cold Spring HarborLaboratory Press, 1988, each incorporated herein it its entirety byreference.

In one embodiment of the present invention, nucleic acids are createdthat encode the antibodies, and that may then be cloned into host cells,expressed and assayed, if desired. Thus, nucleic acids, and particularlyDNA, may be made that encode each protein sequence. These practices arecarried out using well-known procedures. For example, a variety ofmethods that may find use in the present invention are described inMolecular Cloning—A Laboratory Manual, 3^(rd) Ed. (Maniatis, Cold SpringHarbor Laboratory Press, New York, 2001), and Current Protocols inMolecular Biology (John Wiley & Sons), each incorporated herein it itsentirety by reference. As will be appreciated by those skilled in theart, the generation of exact sequences for a library comprising a largenumber of sequences is potentially expensive and time consuming. By“library” herein is meant a set of variants in any form, including butnot limited to a list of nucleic acid or amino acid sequences, a list ofnucleic acid or amino acid substitutions at variable positions, aphysical library comprising nucleic acids that encode the librarysequences, or a physical library comprising the variant proteins, eitherin purified or unpurified form. Accordingly, there are a variety oftechniques that may be used to efficiently generate libraries of thepresent invention. Such methods that may find use in the presentinvention are described or referenced in U.S. Pat. No. 6,403,312; U.S.Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S. Ser. No. 10/218,102;PCT WO 01/40091; and PCT WO 02/25588, each incorporated herein it itsentirety by reference. Such methods include but are not limited to geneassembly methods, PCR-based method and methods which use variations ofPCR, ligase chain reaction-based methods, pooled oligo methods such asthose used in synthetic shuffling, error-prone amplification methods andmethods which use oligos with random mutations, classical site-directedmutagenesis methods, cassette mutagenesis, and other amplification andgene synthesis methods. As is known in the art, there are a variety ofcommercially available kits and methods for gene assembly, mutagenesis,vector subcloning, and the like, and such commercial products find usein the present invention for generating nucleic acids that encodeantibodies.

The antibodies of the present invention may be produced by culturing ahost cell transformed with nucleic acid, e.g., an expression vector,containing nucleic acid encoding the antibodies, under the appropriateconditions to induce or cause expression of the protein. The conditionsappropriate for expression will vary with the choice of the expressionvector and the host cell, and will be easily ascertained by one skilledin the art through routine experimentation. A wide variety ofappropriate host cells may be used, including but not limited tomammalian cells, bacteria, insect cells, and yeast. For example, avariety of cell lines that may find use in the present invention aredescribed in the ATCC® cell line catalog, available from the AmericanType Culture Collection.

In one embodiment, the antibodies are expressed in mammalian expressionsystems, including systems in which the expression constructs areintroduced into the mammalian cells using virus such as retrovirus oradenovirus. Any mammalian cells may be used, e.g., human, mouse, rat,hamster, primate cells, etc. Suitable cells also include known researchcells, including but not limited to Jurkat T cells, NIH3T3, CHO, BHK,COS, HEK293, PER C.6, HeLa, Sp2/0, NSO cells and variants thereof. In analternate embodiment, library proteins are expressed in bacterial cells.Bacterial expression systems are well known in the art, and includeEscherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris,and Streptococcus lividans. In alternate embodiments, antibodies areproduced in insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) oryeast cells (e.g. S. cerevisiae, Pichia, etc). In an alternateembodiment, antibodies are expressed in vitro using cell freetranslation systems. In vitro translation systems derived from bothprokaryotic (e.g. E. coli) and eukaryotic (e.g. wheat germ, rabbitreticulocytes) cells are available and may be chosen based on theexpression levels and functional properties of the protein of interest.For example, as appreciated by those skilled in the art, in vitrotranslation is required for some display technologies, for exampleribosome display. In addition, the antibodies may be produced bychemical synthesis methods. Also transgenic expression systems bothanimal (e.g. cow, sheep or goat milk, embryonated hen's eggs, wholeinsect larvae, etc.) and plant (e.g. corn, tobacco, duckweed, etc.)

The nucleic acids that encode the antibodies of the present inventionmay be incorporated into an expression vector in order to express theprotein. A variety of expression vectors may be utilized for proteinexpression. Expression vectors may comprise self-replicatingextra-chromosomal vectors or vectors which integrate into a host genome.Expression vectors are constructed to be compatible with the host celltype. Thus expression vectors which find use in the present inventioninclude but are not limited to those which enable protein expression inmammalian cells, bacteria, insect cells, yeast, and in in vitro systems.As is known in the art, a variety of expression vectors are available,commercially or otherwise, that may find use in the present inventionfor expressing antibodies.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the antibody, and aretypically appropriate to the host cell used to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used

Antibodies may be operably linked to a fusion partner to enabletargeting of the expressed protein, purification, screening, display,and the like. Fusion partners may be linked to the antibody sequence viaa linker sequence. The linker sequence will generally comprise a smallnumber of amino acids, typically less than ten, although longer linkersmay also be used. Typically, linker sequences are selected to beflexible and resistant to degradation. As will be appreciated by thoseskilled in the art, any of a wide variety of sequences may be used aslinkers. For example, a common linker sequence comprises the amino acidsequence GGGGS. A fusion partner may be a targeting or signal sequencethat directs antibody and any associated fusion partners to a desiredcellular location or to the extracellular media. As is known in the art,certain signaling sequences may target a protein to be either secretedinto the growth media, or into the periplasmic space, located betweenthe inner and outer membrane of the cell. A fusion partner may also be asequence that encodes a peptide or protein that enables purificationand/or screening. Such fusion partners include but are not limited topolyhistidine tags (His-tags) (for example H₆ and H₁₀ or other tags foruse with Immobilized Metal Affinity Chromatography (IMAC) systems (e.g.Ni⁺² affinity columns)), GST fusions, MBP fusions, Strep-tag, the BSPbiotinylation target sequence of the bacterial enzyme BirA, and epitopetags which are targeted by antibodies (for example c-myc tags,flag-tags, and the like). As will be appreciated by those skilled in theart, such tags may be useful for purification, for screening, or both.For example, an antibody may be purified using a His-tag by immobilizingit to a Ni⁺² affinity column, and then after purification the sameHis-tag may be used to immobilize the antibody to a Ni⁺² coated plate toperform an ELISA or other binding assay (as described below). A fusionpartner may enable the use of a selection method to screen antibodies(see below). Fusion partners that enable a variety of selection methodsare well-known in the art, and all of these find use in the presentinvention. For example, by fusing the members of an antibody library tothe gene III protein, phage display can be employed (Kay et al., Phagedisplay of peptides and proteins: a laboratory manual, Academic Press,San Diego, Calif., 1996; Lowman et al., 1991, Biochemistry30:10832-10838; Smith, 1985, Science 228:1315-1317, incorporated hereinit its entirety by reference). Fusion partners may enable antibodies tobe labeled. Alternatively, a fusion partner may bind to a specificsequence on the expression vector, enabling the fusion partner andassociated antibody to be linked covalently or noncovalently with thenucleic acid that encodes them.

The methods of introducing exogenous nucleic acid into host cells arewell known in the art, and will vary with the host cell used. Techniquesinclude but are not limited to dextran-mediated transfection, calciumphosphate precipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection of the DNA into nuclei. In the case of mammaliancells, transfection may be either transient or stable.

In one embodiment, antibodies are purified or isolated after expression.Proteins may be isolated or purified in a variety of ways known to thoseskilled in the art. Standard purification methods includechromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use in the present invention forpurification of antibodies. For example, the bacterial proteins A and Gbind to the Fc region. Likewise, the bacterial protein L binds to theFab region of some antibodies, as of course does the antibody's targetantigen. Purification can often be enabled by a particular fusionpartner. For example, antibodies may be purified using glutathione resinif a GST fusion is employed, Ni⁺² affinity chromatography if a His-tagis employed, or immobilized anti-flag antibody if a flag-tag is used.For general guidance in suitable purification techniques, see, e.g.Protein Purification: Principles and Practice, 3^(rd) Ed., Scopes,Springer-Verlag, NY, 1994, incorporated herein it its entirety byreference. The degree of purification necessary will vary depending onthe screen or use of the antibodies. In some instances no purificationis necessary. For example in one embodiment, if the antibodies aresecreted, screening may take place directly from the media. As is wellknown in the art, some methods of selection do not involve purificationof proteins. Thus, for example, if a library of antibodies is made intoa phage display library, protein purification may not be performed.

In Vitro Experimentation

Antibodies may be screened using a variety of methods, including but notlimited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Automation and high-throughputscreening technologies may be utilized in the screening procedures.Screening may employ the use of a fusion partner or label. The use offusion partners has been discussed above. By “labeled” herein is meantthat the antibodies of the invention have one or more elements,isotopes, or chemical compounds attached to enable the detection in ascreen. In general, labels fall into three classes: a) immune labels,which may be an epitope incorporated as a fusion partner that isrecognized by an antibody, b) isotopic labels, which may be radioactiveor heavy isotopes, and c) small molecule labels, which may includefluorescent and colorimetric dyes, or molecules such as biotin thatenable other labeling methods. Labels may be incorporated into thecompound at any position and may be incorporated in vitro or in vivoduring protein expression.

In one embodiment, the functional and/or biophysical properties ofantibodies are screened in an in vitro assay. In vitro assays may allowa broad dynamic range for screening properties of interest. Propertiesof antibodies that may be screened include but are not limited tostability, solubility, and affinity for Fc ligands, for example FcγRs.Multiple properties may be screened simultaneously or individually.Proteins may be purified or unpurified, depending on the requirements ofthe assay. In one embodiment, the screen is a qualitative orquantitative binding assay for binding of antibodies to a protein ornonprotein molecule that is known or thought to bind the antibody. Inone embodiment, the screen is a binding assay for measuring binding tothe target antigen. In an alternate embodiment, the screen is an assayfor binding of antibodies to an Fc ligand, including but not limited tothe family of FcγRs, the neonatal receptor FcRn, the complement proteinC1q, and the bacterial proteins A and G. The Fc ligands may be from anyorganism, e.g., humans, mice, rats, rabbits, monkeys, etc. Bindingassays can be carried out using a variety of methods known in the art,including but not limited to FRET (Fluorescence Resonance EnergyTransfer) and BRET (Bioluminescence Resonance Energy Transfer)-basedassays, AlphaScreen™ (Amplified Luminescent Proximity HomogeneousAssay), Scintillation Proximity Assay, ELISA (Enzyme-LinkedImmunosorbent Assay), SPR (Surface Plasmon Resonance, also known asBiacore™), isothermal titration calorimetry, differential scanningcalorimetry, gel electrophoresis, and chromatography including gelfiltration. These and other methods may take advantage of some fusionpartner or label of the antibody. Assays may employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels.

The biophysical properties of antibodies, for example stability andsolubility, may be screened using a variety of methods known in the art.Protein stability may be determined by measuring the thermodynamicequilibrium between folded and unfolded states. For example, antibodiesof the present invention may be unfolded using chemical denaturant,heat, or pH, and this transition may be monitored using methodsincluding but not limited to circular dichroism spectroscopy,fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy,calorimetry, and proteolysis. As will be appreciated by those skilled inthe art, the kinetic parameters of the folding and unfolding transitionsmay also be monitored using these and other techniques. The solubilityand overall structural integrity of an antibody may be quantitatively orqualitatively determined using a wide range of methods that are known inthe art. Methods which may find use in the present invention forcharacterizing the biophysical properties of antibodies include gelelectrophoresis, isoelectric focusing, capillary electrophoresis,chromatography such as size exclusion chromatography, ion-exchangechromatography, and reversed-phase high performance liquidchromatography, peptide mapping, oligosaccharide mapping, massspectrometry, ultraviolet absorbance spectroscopy, fluorescencespectroscopy, circular dichroism spectroscopy, isothermal titrationcalorimetry, differential scanning calorimetry, analyticalultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays, microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility may be measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevated temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an antibody could be assayed for itsability to bind target antigen, then exposed to elevated temperature forone or more defined periods of time, then assayed for antigen bindingagain. Because unfolded and aggregated protein is not expected to becapable of binding antigen, the amount of activity remaining provides ameasure of the antibody's stability and solubility.

In one embodiment, the library is screened using one or more cell-basedor in vitro assays. For such assays, antibodies, purified or unpurified,are typically added exogenously such that cells are exposed toindividual variants or groups of variants belonging to a library. Theseassays are typically, but not always, based on the biology of theability of the antibody to bind to antigen and mediate some biochemicalevent, for example effector functions like cellular lysis, phagocytosis,ligand/receptor binding inhibition, inhibition of growth and/orproliferation, apoptosis, etc. Such assays often involve monitoring theresponse of cells to antibody, for example cell survival, cell death,cellular phagocytosis, cell lysis, change in cellular morphology, ortranscriptional activation such as cellular expression of a natural geneor reporter gene. For example, such assays may measure the ability ofantibodies to elicit ADCC, ADCP, or CDC. For some assays additionalcells or components, that is in addition to the target cells, may needto be added, for example serum complement, or effector cells such asperipheral blood monocytes (PBMCs), NK cells, macrophages, and the like.Such additional cells may be from any organism, e.g., humans, mice,rats, rabbits, monkeys, etc. Crosslinked or monomeric antibodies maycause apoptosis of certain cell lines expressing the antibody's targetantigen, or they may mediate attack on target cells by immune cellswhich have been added to the assay. Methods for monitoring cell death orviability are known in the art, and include the use of dyes,fluorophores, immunochemical, cytochemical, and radioactive reagents.For example, caspase assays or annexin-flourconjugates may enableapoptosis to be measured, and uptake or release of radioactivesubstrates (e.g. Chromium-51 release assays) or the metabolic reductionof fluorescent dyes such as alamar blue may enable cell growth,proliferation, or activation to be monitored. In one embodiment, theDELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer, MA) is used.Alternatively, dead or damaged target cells may be monitored bymeasuring the release of one or more natural intracellular proteins, forexample lactate dehydrogenase. Transcriptional activation may also serveas a method for assaying function in cell-based assays. In this case,response may be monitored by assaying for natural genes or proteinswhich may be upregulated or down-regulated, for example the release ofcertain interleukins may be measured, or alternatively readout may bevia a luciferase or GFP-reporter construct. Cell-based assays may alsoinvolve the measure of morphological changes of cells as a response tothe presence of an antibody. Cell types for such assays may beprokaryotic or eukaryotic, and a variety of cell lines that are known inthe art may be employed. Alternatively, cell-based screens are performedusing cells that have been transformed or transfected with nucleic acidsencoding the antibodies.

In vitro assays include but are not limited to binding assays, ADCC,CDC, phagocytosis, cytotoxicity, proliferation, apoptosis, necrosis,cell cycle arrest, peroxide/ozone release, chemotaxis of effector cells,inhibition of such assays by reduced effector function antibodies;ranges of activities such as >100× improvement or >100× reduction,blends of receptor activation and the assay outcomes that are expectedfrom such receptor profiles.

In Vivo Experimentation

The biological properties of the antibodies of the present invention maybe characterized in cell, tissue, and whole organism experiments. As isknown in the art, drugs are often tested in animals, including but notlimited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in orderto measure a drug's efficacy for treatment against a disease or diseasemodel, or to measure a drug's pharmacokinetics, toxicity, and otherproperties. The animals may be referred to as disease models. Withrespect to the antibodies of the present invention, a particularchallenge arises when using animal models to evaluate the potential forin-human efficacy of candidate polypeptides—this is due, at least inpart, to the fact that antibodies that have a specific effect on theaffinity for a human Fc receptor may not have a similar affinity effectwith the orthologous animal receptor. These problems can be furtherexacerbated by the inevitable ambiguities associated with correctassignment of true orthologs (Mechetina et al., Immunogenetics, 200254:463-468, incorporated herein it its entirety by reference), and thefact that some orthologs simply do not exist in the animal (e.g. humanspossess an FcγRIIa whereas mice do not). Therapeutics are often testedin mice, including but not limited to nude mice, SCID mice, xenograftmice, and transgenic mice (including knockins and knockouts). Forexample, an antibody of the present invention that is intended as ananti-cancer therapeutic may be tested in a mouse cancer model, forexample a xenograft mouse. In this method, a tumor or tumor cell line isgrafted onto or injected into a mouse, and subsequently the mouse istreated with the therapeutic to determine the ability of the antibody toreduce or inhibit cancer growth and metastasis. An alternative approachis the use of a SCID murine model in which immune-deficient mice areinjected with human Periferal Blood Lymphocytes (PBLs), conferring asemi-functional and human immune system—with an appropriate array ofhuman FcRs—to the mice that have subsequently been injected withantibodies or Fc-polypeptides that target injected human tumor cells. Insuch a model, the Fc-polypeptides that target the desired antigen (suchas her2/neu on SkOV3 ovarian cancer cells) interact with human PBLswithin the mice to engage tumoricidal effector functions. Suchexperimentation may provide meaningful data for determination of thepotential of the antibody to be used as a therapeutic. Any organism,e.g., mammals, may be used for testing. For example because of theirgenetic similarity to humans, monkeys can be suitable therapeuticmodels, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the antibodies of the presentinvention. Tests of the antibodies of the present invention in humansare ultimately required for approval as drugs, and thus of course theseexperiments are contemplated. Thus the antibodies of the presentinvention may be tested in humans to determine their therapeuticefficacy, toxicity, pharmacokinetics, and/or other clinical properties.

The antibodies of the present invention may confer superior performanceon Fc-containing therapeutics in animal models or in humans. Thereceptor binding profiles of such antibodies, as described in thisspecification, may, for example, be selected to increase the potency ofcytotoxic drugs or to target specific effector functions or effectorcells to improve the selectivity of the drug's action. Further, receptorbinding profiles can be selected that may reduce some or all effectorfunctions thereby reducing the side-effects or toxicity of suchFc-containing drug. For example, an antibody with reduced binding toFcγRIIIa, FcγRI and FcγRIIa can be selected to eliminate mostcell-mediated effector function, or an antibody with reduced binding toC1q may be selected to limit complement-mediated effector functions. Insome contexts, such effector functions are known to have potential toxiceffects, therefore eliminating them may increase the safety of theFc-bearing drug and such improved safety may be characterized in animalmodels. In some contexts, such effector functions are known to mediatethe desirable therapeutic activity, therefore enhancing them mayincrease the activity or potency of the Fc-bearing drug and suchimproved activity or potency may be characterized in animal models.

Optimized antibodies can be tested in a variety of orthotopic tumormodels. These clinically relevant animal models are important in thestudy of pathophysiology and therapy of aggressive cancers likepancreatic, prostate and breast cancer. Immune deprived mice including,but not limited to athymic nude or SCID mice are frequently used inscoring of local and systemic tumor spread from the site of intraorgan(e.g. pancreas, prostate or mammary gland) injection of human tumorcells or fragments of donor patients.

In some embodiments, antibodies of the present invention may be assessedfor efficacy in clinically relevant animal models of various humandiseases. In many cases, relevant models include various transgenicanimals for specific tumor antigens.

Relevant transgenic models such as those that express human Fc receptors(e.g., CD16 including the gamma chain, FcγR1, RIIa/b, and others) couldbe used to evaluate and test antibodies and Fc-fusions in theirefficacy. The evaluation of antibodies by the introduction of humangenes that directly or indirectly mediate effector function in mice orother rodents may enable physiological studies of efficacy in tumortoxicity or other diseases such as autoimmune disorders and RA. Human Fcreceptors such as FcγRIIIa may possess polymorphisms such as that inposition 158 V or F which would further enable the introduction ofspecific and combinations of human polymorphisms into rodents. Thevarious studies involving polymorphism-specific FcRs are not limited tothis section, however, and encompasses all discussions and applicationsof FcRs in general as specified in throughout this application.antibodies of the present invention may confer superior activity onFc-containing drugs in such transgenic models, in particular variantswith binding profiles optimized for human FcγRIIIa mediated activity mayshow superior activity in transgenic CD16 mice. Similar improvements inefficacy in mice transgenic for the other human Fc receptors, e.g.FcγRIIa, FcγRI, etc., may be observed for antibodies with bindingprofiles optimized for the respective receptors. Mice transgenic formultiple human receptors would show improved activity for antibodieswith binding profiles optimized for the corresponding multiplereceptors, for example as outlined in FIG. 3.

Because of the difficulties and ambiguities associated with using animalmodels to characterize the potential efficacy of candidate therapeuticantibodies in a human patient, some variant polypeptides of the presentinvention may find utility as proxies for assessing potential in-humanefficacy. Such proxy molecules may mimic—in the animal system—the FcRand/or complement biology of a corresponding candidate human antibody.This mimicry is most likely to be manifested by relative associationaffinities between specific antibodies and animal vs. human receptors.For example, if one were using a mouse model to assess the potentialin-human efficacy of an antibody that has enhanced affinity for humanFcγRIIIa, an appropriate proxy variant would have enhanced affinity formouse FcγRIII-2 (mouse CD16-2). Alternatively if one were using a mousemodel to assess the potential in-human efficacy of an antibody that hasreduced affinity for the inhibitory human FcγRIIb, an appropriate proxyvariant would have reduced affinity for mouse FcγRII. It should also benoted that the proxy antibodies could be created in the context of ahuman antibody, an animal antibody, or both.

In one embodiment, the testing of antibodies may include study ofefficacy in primates (e.g. cynomolgus monkey model) to facilitate theevaluation of depletion of specific target cells harboring targetantigen. Additional primate models include but are not limited to thatof the rhesus monkey and Fc polypeptides in therapeutic studies ofautoimmune, transplantation, and cancer.

Toxicity studies are performed to determine the antibody or Fc-fusionrelated-effects that cannot be evaluated in standard pharmacologyprofile or occur only after repeated administration of the agent. Mosttoxicity tests are performed in two species—a rodent and a non-rodent—toensure that any unexpected adverse effects are not overlooked before newtherapeutic entities are introduced into man. In general, these modelsmay measure a variety of toxicities including genotoxicity, chronictoxicity, immunogenicity, reproductive/developmental toxicity, andcarcinogenicity. Included within the aforementioned parameters arestandard measurement of food consumption, bodyweight, antibodyformation, clinical chemistry, and macro- and microscopic examination ofstandard organs/tissues (e.g. cardiotoxicity). Additional parameters ofmeasurement are injection site trauma and the measurement ofneutralizing antibodies, if any. Traditionally, monoclonal antibodytherapeutics, naked or conjugated are evaluated for cross-reactivitywith normal tissues, immunogenicity/antibody production, conjugate orlinker toxicity and “bystander” toxicity of radiolabeled species.Nonetheless, such studies may have to be individualized to addressspecific concerns and following the guidance set by ICH S6 (Safetystudies for biotechnological products also noted above). As such, thegeneral principles are that the products are sufficiently wellcharacterized and for which impurities/contaminants have been removed,that the test material is comparable throughout development, and GLPcompliance.

The pharmacokinetics (PK) of the antibodies of the invention can bestudied in a variety of animal systems, with the most relevant beingnon-human primates such as the cynomolgus, rhesus monkeys. Single orrepeated i.v./s.c. administrations over a dose range of 6000-fold(0.05-300 mg/kg) can be evaluated for the half-life (days to weeks)using plasma concentration and clearance as well as volume ofdistribution at a steady state and level of systemic absorbance can bemeasured. Examples of such parameters of measurement generally includemaximum observed plasma concentration (Cmax), the time to reach Cmax(Tmax), the area under the plasma concentration-time curve from time 0to infinity [AUC(0-inf] and apparent elimination half-life (T₁₁₂).Additional measured parameters could include compartmental analysis ofconcentration-time data obtained following i.v. administration andbioavailability. Examples of pharmacological/toxicological studies usingcynomolgus have been established for Rituxan® and Zevalin® in whichmonoclonal antibodies to CD20 are cross-reactive. Biodistribution,dosimetry (for radiolabled antibodies), and PK studies can also be donein rodent models. Such studies would evaluate tolerance at all dosesadministered, toxicity to local tissues, localization to rodentxenograft animal models, depletion of target cells (e.g. CD20 positivecells).

The antibodies of the present invention may confer superiorpharmacokinetics on Fc-containing therapeutics in animal systems or inhumans. For example, increased binding to FcRn may increase thehalf-life and exposure of the Fc-containing drug. Alternatively,decreased binding to FcRn may decrease the half-life and exposure of theFc-containing drug in cases where reduced exposure is favorable such aswhen such drug has side-effects.

It is known in the art that the array of Fc receptors is differentiallyexpressed on various immune cell types, as well as in different tissues.Differential tissue distribution of Fc receptors may ultimately have animpact on the pharmacodynamic (PD) and pharmacokinetic (PK) propertiesof antibodies of the present invention. Because antibodies of thepresentation have varying affinities for the array of Fc receptors,further screening of the polypeptides for PD and/or PK properties may beextremely useful for defining the optimal balance of PD, PK, andtherapeutic efficacy conferred by each candidate polypeptide.

Pharmacodynamic studies may include, but are not limited to, targetingspecific tumor cells or blocking signaling mechanisms, measuringdepletion of target antigen expressing cells or signals, etc. Theantibodies of the present invention may target particular effector cellpopulations and thereby direct Fc-containing drugs to recruit certainactivities to improve potency or to increase penetration into aparticularly favorable physiological compartment. For example,neutrophil activity and localization can be targeted, e.g., by anantibody that targets FcγRIIIb. Such pharmacodynamic effects may bedemonstrated in animal models or in humans.

Clinical Use

The antibodies of the present invention may be used for varioustherapeutic purposes. As will be appreciated by those in the art, theantibodies of the present invention may be used for any therapeuticpurpose that uses antibodies and the like. In one embodiment, theantibodies are administered to a patient to treat disorders includingbut not limited to cancer, autoimmune and inflammatory diseases, andinfectious diseases.

A “patient” for the purposes of the present invention includes bothhumans and other animals, e.g., mammals, e.g., humans. Thus theantibodies of the present invention have both human therapy andveterinary applications. The term “treatment” or “treating” in thepresent invention is meant to include therapeutic treatment, as well asprophylactic, or suppressive measures for a disease or disorder. Thus,for example, successful administration of an antibody prior to onset ofthe disease results in treatment of the disease. As another example,successful administration of an optimized antibody after clinicalmanifestation of the disease to combat the symptoms of the diseasecomprises treatment of the disease. “Treatment” and “treating” alsoencompasses administration of an optimized antibody after the appearanceof the disease in order to eradicate the disease. Successfuladministration of an agent after onset and after clinical symptoms havedeveloped, with possible abatement of clinical symptoms and perhapsamelioration of the disease, comprises treatment of the disease. Those“in need of treatment” include mammals already having the disease ordisorder, as well as those prone to having the disease or disorder,including those in which the disease or disorder is to be prevented.

In one embodiment, an antibody of the present invention is administeredto a patient having a disease involving inappropriate expression of aprotein or other molecule. Within the scope of the present inventionthis is meant to include diseases and disorders characterized byaberrant proteins, due for example to alterations in the amount of aprotein present, protein localization, posttranslational modification,conformational state, the presence of a mutant or pathogen protein, etc.Similarly, the disease or disorder may be characterized by alterationsmolecules including but not limited to polysaccharides and gangliosides.An overabundance may be due to any cause, including but not limited tooverexpression at the molecular level, prolonged or accumulatedappearance at the site of action, or increased activity of a proteinrelative to normal. Included within this definition are diseases anddisorders characterized by a reduction of a protein. This reduction maybe due to any cause, including but not limited to reduced expression atthe molecular level, shortened or reduced appearance at the site ofaction, mutant forms of a protein, or decreased activity of a proteinrelative to normal. Such an overabundance or reduction of a protein canbe measured relative to normal expression, appearance, or activity of aprotein, and the measurement may play an important role in thedevelopment and/or clinical testing of the antibodies of the presentinvention.

By “cancer” and “cancerous” herein refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma),neuroendocrine tumors, mesothelioma, schwanoma, meningioma,adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

More particular examples of such cancers include hematologicmalignancies, such as non-Hodgkin's lymphomas (NHL). NHL cancers includebut are not limited to Burkitt's lymphoma (BL), small lymphocyticlymphoma/chronic lymphocytic leukemia (SLL/CLL), mantle cell lymphoma(MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLCL),marginal zone lymphoma (MZL), hairy cell leukemia (HCL) andlymphoplasmacytic leukemia (LPL), extranodal marginal zone B-celllymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginalzone B cell lymphoma, mediastinal large cell lymphoma, intravascularlarge cell lymphoma, primary effusion lymphoma, precursorB-lymphoblastic leukemia/lymphoma, precursor T- and NK-cells lymphoma(precursor T lymphoblastic lymphoma, blastic NK cell lymphoma), tumorsof the mature T and NK cells, including peripheral T-cell lymphoma andleukemia (PTL), adult T-cell leukemia/T-cell lymphomas and largegranular lymphocytic leukemia, T-cell chronic lymphocyticleukemia/prolymphocytic leukemia, T-cell large granular lymphocyticleukemia, aggressive NK-cell leukemia, extranodal T-/NK cell lymphoma,enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma,anaplastic large cell lymphoma (ALCL), angiocetric andangioimmunoblastic T-cell lymphoma, mycosis fungoides/Sezary syndrome,and cutaneous T-cell lymphoma (CTCL). Other cancers that may betreatable by the antibodies of the invention include but are not limitedto Hodgkin's lymphoma, tumors of lymphocyte precursor cells, includingB-cell acute lymphoblastic leukemia/lymphoma (B-ALL), and T-cell acutelymphoblastic leukemia/lymphoma (T-ALL), thymoma, Langerhans cellhistocytosis, multiple myeloma (MM), myeloid neoplasias such as acutemyelogenous leukemias (AML), including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders (MDS), including chronicmyelogenous leukemia (CML). Other cancers that may be treatable by theantibodies of the invention include but are not limited to tumors of thecentral nervous system such as glioma, glioblastoma, neuroblastoma,astrocytoma, medulloblastoma, ependymoma, and retinoblastoma; solidtumors of the head and neck (e.g. nasopharyngeal cancer, salivary glandcarcinoma, and esophageal cancer), lung (eg. small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung and squamouscarcinoma of the lung), digestive system (eg. gastric or stomach cancerincluding gastrointestinal cancer, cancer of the bile duct or biliarytract, colon cancer, rectal cancer, colorectal cancer, and analcarcinoma), reproductive system (eg. testicular, penile, or prostatecancer, uterine, vaginal, vulval, cervical, ovarian, and endometrialcancer), skin (eg. melanoma, basal cell carcinoma, squamous cell cancer,actinic keratosis), liver (eg. liver cancer, hepatic carcinoma,hepatocellular cancer, and hepatoma), bone (eg. osteoclastoma, andosteolytic bone cancers) additional tissues and organs (eg. pancreaticcancer, bladder cancer, kidney or renal cancer, thyroid cancer, breastcancer, cancer of the peritoneum, and Kaposi's sarcoma), and tumors ofthe vascular system (eg. angiosarcoma and hemagiopericytoma).

Indications that may be treated by anti-CD40 antibodies of the inventioninclude but are not limited to all non-Hodgkin's lymphomas (NHL),especially refractory/resistant NHL, chronic lymphocytic leukemia (CLL),B-cell acute lymphoblastic leukemia/lymphoma (B-ALL), mantle celllymphoma (MCL), and multiple myeloma (MM).

Autoimmunity results from a breakdown of self-tolerance involvinghumoral and/or cell-mediated immune mechanisms. Among the consequencesof failure in central and/or peripheral tolerance are survival andactivation of self-reactive B cells and T cells. Several autoimmunediseases are defined by excessive activation of both B and/or Tlymphocytes. Activation of these cells requires in cooperation, antigenengagement and co-stimulatory signals from interacting lymphocytes. Thusantibody-mediated depletion, inhibition, anti-proliferation, and/orblockade of B cells and/or T cells are therapeutic approaches for thetreatment of autoimmune disease.

By “autoimmune diseases” herein include allogenic islet graft rejection,alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies(ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune myocarditis, autoimmuneneutropenia, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, autoimmune urticaria, Behcet's disease, bullouspemphigoid, cardiomyopathy, Castleman's syndrome, celiacspruce-dermatitis, chronic fatigue immune dysfunction syndrome, chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn'sdisease, dermatomyositis, discoid lupus, essential mixedcryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis,glomerulonephritis, Grave's disease, Guillain-Barre, Goodpasture'ssyndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis,hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, IgM polyneuropathies, immune mediatedthrombocytopenia, juvenile arthritis, Kawasaki's disease, lichenplantus, lupus erythematosus, Meniere's disease, mixed connective tissuedisease, multiple sclerosis, type 1 diabetes mellitus, myastheniagravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobinulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Reynauld'sphenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis,scleroderma, Sjorgen's syndrome, solid organ transplant rejection,stiff-man syndrome, systemic lupus erythematosus, takayasu arteritis,temporal arteristis/giant cell arteritis, thrombotic thrombocytopeniapurpura, ulcerative colitis, uveitis, vasculitides such as dermatitisherpetiformis vasculitis, vitiligo, and Wegner's granulomatosis.

By “inflammatory disorders” herein include acute respiratory distresssyndrome (ARDS), acute septic arthritis, adjuvant arthritis, juvenileidiopathic arthritis, allergic encephalomyelitis, allergic rhinitis,allergic vasculitis, allergy, asthma, atherosclerosis, chronicinflammation due to chronic bacterial or viral infections, chronicobstructive pulmonary disease (COPD), coronary artery disease,encephalitis, inflammatory bowel disease, inflammatory osteolysis,inflammation associated with acute and delayed hypersensitivityreactions, inflammation associated with tumors, peripheral nerve injuryor demyelinating diseases, inflammation associated with tissue traumasuch as burns and ischemia, inflammation due to meningitis, multipleorgan injury syndrome, pulmonary fibrosis, sepsis and septic shock,Stevens-Johnson syndrome, undifferentiated arthropy, andundifferentiated spondyloarthropathy.

By “infectious diseases” herein include diseases caused by pathogenssuch as viruses, bacteria, fungi, protozoa, and parasites. Infectiousdiseases may be caused by viruses including adenovirus, cytomegalovirus,dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C,herpes simplex type I, herpes simplex type II, human immunodeficiencyvirus (HIV), human papilloma virus (HPV), influenza, measles, mumps,papova virus, polio, respiratory syncytial virus (RSV), rinderpest,rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis,and the like. Infections diseases may also be caused by bacteriaincluding Bacillus anthracis, Borrelia burgdorferi, Campylobacterjejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridiumtetani, Diptheria, E. coli, Legionella, Helicobacter pylori,Mycobacterium rickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonasaeruginosa, S. pneumonia, Streptococcus, Staphylococcus, Vibriacholerae, Yersinia pestis, and the like. Infectious diseases may also becaused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis,Candida albicans, Coccidioides immitis, Cryptococcus neoformans,Histoplasma capsulatum, Penicillium marneffei, and the like. Infectiousdiseases may also be caused by protozoa and parasites such as chlamydia,kokzidioa, leishmania, malaria, rickettsia, trypanosoma, and the like.

Furthermore, antibodies of the present invention may be used to preventor treat additional conditions including but not limited to heartconditions such as congestive heart failure (CHF), myocarditis and otherconditions of the myocardium; skin conditions such as rosecea, acne, andeczema; bone and tooth conditions such as bone loss, osteoporosis,Paget's disease, Langerhans' cell histiocytosis, periodontal disease,disuse osteopenia, osteomalacia, monostotic fibrous dysplasia,polyostotic fibrous dysplasia, bone metastasis, bone pain management,humoral malignant hypercalcemia, periodontal reconstruction, spinal cordinjury, and bone fractures; metabolic conditions such as Gaucher'sdisease; endocrine conditions such as Cushing's syndrome; andneurological conditions.

A number of the receptors that may interact with the antibodies of thepresent invention are polymorphic in the human population. For a givenpatient or population of patients, the efficacy of the antibodies of thepresent invention may be affected by the presence or absence of specificpolymorphisms in proteins. For example, FcγRIIIA is polymorphic atposition 158, which is commonly either V (high affinity) or F (lowaffinity). Patients with the V/V homozygous genotype are observed tohave a better clinical response to treatment with the anti-CD20 antibodyRituxan® (rituximab), likely because these patients mount a stronger NKresponse (Dall'Ozzo et al. (2004) Cancer Res. 64:4664-9, incorporatedherein it its entirety by reference). Additional polymorphisms includebut are not limited to FcγRIIA R131 or H131, and such polymorphisms areknown to either increase or decrease Fc binding and subsequentbiological activity, depending on the polymorphism. antibodies of thepresent invention may bind to a particular polymorphic form of areceptor, for example FcγRIIIA 158 V, or to bind with equivalentaffinity to all of the polymorphisms at a particular position in thereceptor, for example both the 158V and 158F polymorphisms of FcγRIIIA.In one embodiment, antibodies of the present invention may haveequivalent binding to polymorphisms that may be used in an antibody toeliminate the differential efficacy seen in patients with differentpolymorphisms. Such a property may give greater consistency intherapeutic response and reduce non-responding patient populations. Suchvariant Fc with identical binding to receptor polymorphisms may haveincreased biological activity, such as ADCC, CDC or circulatinghalf-life, or alternatively decreased activity, via modulation of thebinding to the relevant Fc receptors. In one embodiment, antibodies ofthe present invention may bind with higher or lower affinity to one ofthe polymorphisms of a receptor, either accentuating the existingdifference in binding or reversing the difference. Such a property mayallow creation of therapeutics particularly tailored for efficacy with apatient population possessing such polymorphism. For example, a patientpopulation possessing a polymorphism with a higher affinity for aninhibitory receptor such as FcγRIIB could receive a drug containing anantibody with reduced binding to such polymorphic form of the receptor,creating a more efficacious drug.

In one embodiment, patients are screened for one or more polymorphismsin order to predict the efficacy of the antibodies of the presentinvention. This information may be used, for example, to select patientsto include or exclude from clinical trials or, post-approval, to provideguidance to physicians and patients regarding appropriate dosages andtreatment options. For example, in patients that are homozygous orheterozygous for FcγRIIIA 158F antibody drugs such as the anti-CD20 mAb,Rituxan® are minimally effective (Carton 2002 Blood 99: 754-758; Weng2003 J. Clin. Oncol. 21:3940-3947, each incorporated herein it itsentirety by reference); such patients may show a much better clinicalresponse to the antibodies of the present invention. In one embodiment,patients are selected for inclusion in clinical trials for an antibodyof the present invention if their genotype indicates that they arelikely to respond significantly better to an antibody of the presentinvention as compared to one or more currently used antibodytherapeutics. In another embodiment, appropriate dosages and treatmentregimens are determined using such genotype information. In anotherembodiment, patients are selected for inclusion in a clinical trial orfor receipt of therapy post-approval based on their polymorphismgenotype, where such therapy contains an antibody engineered to bespecifically efficacious for such population, or alternatively wheresuch therapy contains an antibody that does not show differentialactivity to the different forms of the polymorphism.

Included in the present invention are diagnostic tests to identifypatients who are likely to show a favorable clinical response to anantibody of the present invention, or who are likely to exhibit asignificantly better response when treated with an antibody of thepresent invention versus one or more currently used antibodytherapeutics. Any of a number of methods for determining FcγRpolymorphisms in humans known in the art may be used.

Furthermore, the present invention comprises prognostic tests performedon clinical samples such as blood and tissue samples. Such tests mayassay for effector function activity, including but not limited to ADCC,CDC, phagocytosis, and opsonization, or for killing, regardless ofmechanism, of cancerous or otherwise pathogenic cells. In oneembodiment, ADCC assays, such as those described previously, are used topredict, for a specific patient, the efficacy of a given antibody of thepresent invention. Such information may be used to identify patients forinclusion or exclusion in clinical trials, or to inform decisionsregarding appropriate dosages and treatment regimens. Such informationmay also be used to select a drug that contains a particular antibodythat shows superior activity in such assay.

Formulation

Pharmaceutical compositions are contemplated wherein an antibody of thepresent invention and one or more therapeutically active agents areformulated. Formulations of the antibodies of the present invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed., 1980, incorporated herein it its entirety by reference), in theform of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, acetate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; sweeteners and other flavoring agents;fillers such as microcrystalline cellulose, lactose, corn and otherstarches; binding agents; additives; coloring agents; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). In one embodiment, the pharmaceuticalcomposition that comprises the antibody of the present invention may bein a water-soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly useful are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. The formulations to beused for in vivo administration should be sterile. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods.

The antibodies disclosed herein may also be formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids and/or surfactant that is useful for deliveryof a therapeutic agent to a mammal. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al., 1980, ProcNatl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No.4,544,545; and PCT WO 97/38731, each incorporated herein it its entiretyby reference. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556, incorporated herein it its entirety byreference. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes. Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. A chemotherapeutic agent or other therapeutically active agentis optionally contained within the liposome (Gabizon et al., 1989, JNational Cancer Inst 81:1484, incorporated herein it its entirety byreference).

The antibody and other therapeutically active agents may also beentrapped in microcapsules prepared by methods including but not limitedto coacervation techniques, interfacial polymerization (for exampleusing hydroxymethylcellulose or gelatin-microcapsules, orpoly-(methylmethacylate) microcapsules), colloidal drug delivery systems(for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules), and macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980, incorporated herein it its entirety by reference.Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymer, which matrices are in the form of shaped articles,e.g. films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for examplepoly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919, incorporated herein it its entirety byreference), copolymers of L-glutamic acid and gamma ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the Lupron Depot® (which are injectablemicrospheres composed of lactic acid-glycolic acid copolymer andleuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, and ProLease®(commercially available from Alkermes), which is a microsphere-baseddelivery system composed of the desired bioactive molecule incorporatedinto a matrix of poly-DL-lactide-co-glycolide (PLG).

Administration

Administration of the pharmaceutical composition comprising an antibodyof the present invention, e.g., in the form of a sterile aqueoussolution, may be done in a variety of ways, including, but not limitedto orally, subcutaneously, intravenously, intranasally, intraotically,transdermally, topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary, vaginally,parenterally, rectally, or intraocularly. In some instances, for examplefor the treatment of wounds, inflammation, etc., the antibody may bedirectly applied as a solution or spray. As is known in the art, thepharmaceutical composition may be formulated accordingly depending uponthe manner of introduction.

Subcutaneous administration may be desirable in some circumstancesbecause the patient may self-administer the pharmaceutical composition.Many protein therapeutics are not sufficiently potent to allow forformulation of a therapeutically effective dose in the maximumacceptable volume for subcutaneous administration. This problem may beaddressed in part by the use of protein formulations comprisingarginine-HCl, histidine, and polysorbate (see WO 04091658, incorporatedherein it its entirety by reference). Antibodies of the presentinvention may be more amenable to subcutaneous administration due to,for example, increased potency, improved serum half-life, or enhancedsolubility.

As is known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The antibodies of the present invention may also bedelivered using such methods. For example, administration may be byintravenous infusion with 0.9% sodium chloride as an infusion vehicle.

Pulmonary delivery may be accomplished using an inhaler or nebulizer anda formulation comprising an aerosolizing agent. For example, AERx®inhalable technology commercially available from Aradigm, or Inhance™pulmonary delivery system commercially available from NektarTherapeutics may be used. Antibodies of the present invention may bemore amenable to intrapulmonary delivery. FcRn is present in the lung,and may promote transport from the lung to the bloodstream (e.g.Syntonix WO 04004798, Bitonti et al. (2004) Proc. Nat. Acad. Sci.101:9763-8, each incorporated herein it its entirety by reference).Accordingly, antibodies that bind FcRn more effectively in the lung orthat are released more efficiently in the bloodstream may have improvedbioavailability following intrapulmonary administration. Antibodies ofthe present invention may also be more amenable to intrapulmonaryadministration due to, for example, improved solubility or alteredisoelectric point.

Furthermore, antibodies of the present invention may be more amenable tooral delivery due to, for example, improved stability at gastric pH andincreased resistance to proteolysis. Furthermore, FcRn appears to beexpressed in the intestinal epithelia of adults (Dickinson et al. (1999)J. Clin. Invest. 104:903-11, incorporated herein it its entirety byreference), so antibodies of the present invention with improved FcRninteraction profiles may show enhanced bioavailability following oraladministration. FcRn mediated transport of antibodies may also occur atother mucus membranes such as those in the gastrointestinal,respiratory, and genital tracts (Yoshida et al. (2004) Immunity20:769-83, incorporated herein it its entirety by reference).

In addition, any of a number of delivery systems are known in the artand may be used to administer the antibodies of the present invention.Examples include, but are not limited to, encapsulation in liposomes,microparticles, microspheres (eg. PLA/PGA microspheres), and the like.Alternatively, an implant of a porous, non-porous, or gelatinousmaterial, including membranes or fibers, may be used. Sustained releasesystems may comprise a polymeric material or matrix such as polyesters,hydrogels, poly(vinylalcohol),polylactides, copolymers of L-glutamicacid and ethyl-L-glutamate, ethylene-vinyl acetate, lactic acid-glycolicacid copolymers such as the Lupron Depot®, andpoly-D-(−)-3-hydroxyburyric acid. It is also possible to administer anucleic acid encoding the antibody of the current invention, for exampleby retroviral infection, direct injection, or coating with lipids, cellsurface receptors, or other transfection agents. In all cases,controlled release systems may be used to release the antibody at orclose to the desired location of action.

Dosing

The dosing amounts and frequencies of administration are, in oneembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the therapeutically active antibody in theformulation may vary from about 0.1 to 100 weight %. In one embodiment,the concentration of the antibody is in the range of 0.003 μM to 1.0molar. In order to treat a patient, a therapeutically effective dose ofthe antibody of the present invention may be administered. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. Dosages may range from 0.0001 to 100mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg ofbody weight, e.g., 1 to 10 mg/kg of body weight.

In some embodiments, only a single dose of the antibody is used. Inother embodiments, multiple doses of the antibody are administered. Theelapsed time between administrations may be less than 1 hour, about 1hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours,about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.

In other embodiments the antibodies of the present invention areadministered in metronomic dosing regimes, either by continuous infusionor frequent administration without extended rest periods. Suchmetronomic administration may involve dosing at constant intervalswithout rest periods. Typically such regimens encompass chronic low-doseor continuous infusion for an extended period of time, for example 1-2days, 1-2 weeks, 1-2 months, or up to 6 months or more. The use of lowerdoses may minimize side effects and the need for rest periods.

In certain embodiments the antibody of the present invention and one ormore other prophylactic or therapeutic agents are cyclicallyadministered to the patient. Cycling therapy involves administration ofa first agent at one time, a second agent at a second time, optionallyadditional agents at additional times, optionally a rest period, andthen repeating this sequence of administration one or more times. Thenumber of cycles is typically from 2-10. Cycling therapy may reduce thedevelopment of resistance to one or more agents, may minimize sideeffects, or may improve treatment efficacy.

Combination Therapies

The antibodies of the present invention may be administeredconcomitantly with one or more other therapeutic regimens or agents. Theadditional therapeutic regimes or agents may be used to improve theefficacy or safety of the antibody. Also, the additional therapeuticregimes or agents may be used to treat the same disease or a comorbidityrather than to alter the action of the antibody. For example, anantibody of the present invention may be administered to the patientalong with chemotherapy, radiation therapy, or both chemotherapy andradiation therapy. The antibody of the present invention may beadministered in combination with one or more other prophylactic ortherapeutic agents, including but not limited to cytotoxic agents,chemotherapeutic agents, cytokines, growth inhibitory agents,anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,cardioprotectants, immunostimulatory agents, immunosuppressive agents,agents that promote proliferation of hematological cells, angiogenesisinhibitors, protein tyrosine kinase (PTK) inhibitors, additionalantibodies, FcγRIIb or other Fc receptor inhibitors, or othertherapeutic agents.

The terms “in combination with” and “co-administration” are not limitedto the administration of the prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the antibody of thepresent invention and the other agent or agents are administered in asequence and within a time interval such that they may act together toprovide a benefit that is increased versus treatment with only eitherthe antibody of the present invention or the other agent or agents. Inone embodiment, that the antibody and the other agent or agents actadditively, e.g., they act synergistically. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended. The skilled medical practitioner can determine empirically, orby considering the pharmacokinetics and modes of action of the agents,the appropriate dose or doses of each therapeutic agent, as well as theappropriate timings and methods of administration.

In one embodiment, the antibodies of the present invention areadministered with one or more additional molecules comprising antibodiesor Fc. The antibodies of the present invention may be co-administeredwith one or more other antibodies that have efficacy in treating thesame disease or an additional comorbidity; for example two antibodiesmay be administered that recognize two antigens that are overexpressedin a given type of cancer, or two antigens that mediate pathogenesis ofan autoimmune or infectious disease.

Examples of anti-cancer antibodies that may be co-administered include,but are not limited to, anti-17-1A cell surface antigen antibodies suchas Panorex™ (edrecolomab); anti-4-1 BB antibodies; anti-4Dc antibodies;anti-A33 antibodies such as A33 and CDP-833; anti-αVβ1 integrinantibodies such as natalizumab; anti-αVβ7 integrin antibodies such asLDP-02; anti-αVβ1 integrin antibodies such as F-200, M-200, and SJ-749;anti-αVβ3 integrin antibodies such as abciximab, CNTO-95, Mab-17E6, andVitaxin™; anti-complement factor 5 (C5) antibodies such as 5G1.1;anti-CA125 antibodies such as OvaRex® (oregovomab); anti-CD3 antibodiessuch as Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies such asIDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies such as Oncolysin B andOncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodiessuch as B43, MT-103, and Oncolysin B; anti-CD20 antibodies such as 2H7,2H7.v16, 2H7.v114, 2H7.v115, Bexxar® (tositumomab, I-131 labeledanti-CD20), Rituxan® (rituximab), and Zevalin® (Ibritumomab tiuxetan,Y-90 labeled anti-CD20); anti-CD22 antibodies such as Lymphocide™(epratuzumab, Y-90 labeled anti-CD22); anti-CD23 antibodies such asIDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax®(daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30;anti-CD33 antibodies such as Mylotarg® (gemtuzumab ozogamicin),Oncolysin M, and Smart M195; anti-CD38 antibodies; anti-CD40 antibodiessuch as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8,Antova™, and IDEC-131; anti-CD44 antibodies such as bivatuzumab;anti-CD46 antibodies; anti-CD402 antibodies such as Campath®(alemtuzumab); anti-CD405 antibodies such as SC-1; anti-CD406 antibodiessuch as huN901-DM1; anti-CD64 antibodies such as MDX-33; anti-CD66eantibodies such as XR-303; anti-CD74 antibodies such as IMMU-110;anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89antibodies such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodiessuch as B-B4-DM1; anti-CD146 antibodies such as AA-98; anti-CD148antibodies; anti-CEA antibodies such as cT84.66, labetuzumab, andPentacea™; anti-CTLA-4 antibodies such as MDX-101; anti-CXCR4antibodies; anti-EGFR antibodies such as ABX-EGF, Erbitux® (cetuximab),IMC-C225, and Merck Mab 425; anti-EpCAM antibodies such as Crucell'santi-EpCAM, ING-1, and IS-IL-2; anti-ephrin B2/EphB4 antibodies;anti-Her2 antibodies such as Herceptin®, MDX-210; anti-FAP (fibroblastactivation protein) antibodies such as sibrotuzumab; anti-ferritinantibodies such as NXT-211; anti-FGF-1 antibodies; anti-FGF-3antibodies; anti-FGF-8 antibodies; anti-FGFR antibodies, anti-fibrinantibodies; anti-G250 antibodies such as WX-G250 and Rencarex®; anti-GD2ganglioside antibodies such as EMD-273063 and TriGem; anti-GD3ganglioside antibodies such as BEC2, KW-2871, and mitumomab;anti-gpIIb/IIIa antibodies such as ReoPro; anti-heparinase antibodies;anti-Her2/ErbB2 antibodies such as Herceptin® (trastuzumab), MDX-210,and pertuzumab; anti-HLA antibodies such as Oncolym®, Smart 1D10;anti-HM1.24 antibodies; anti-ICAM antibodies such as ICM3; anti-IgAreceptor antibodies; anti-IGF-1 antibodies such as CP-751871 and EM-164;anti-IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodies such asCNTO-328 and elsilimomab; anti-IL-15 antibodies such as HuMax™-IL15;anti-KDR antibodies; anti-laminin 5 antibodies; anti-Lewis Y antigenantibodies such as Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Muc1antibodies such as BravaRex and TriAb; anti-NCAM antibodies such asERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn andTherex; anti-PSA antibodies; anti-PSCA antibodies such as IG8; anti-Ptkantibodies; anti-PTN antibodies; anti-RANKL antibodies such as AMG-162;anti-RLIP76 antibodies; anti-SK-1 antigen antibodies such as MonopharmC; anti-STEAP antibodies; anti-TAG72 antibodies such as CC49-SCA andMDX-220; anti-TGF-β antibodies such as CAT-152; anti-TNF-α antibodiessuch as CDP571, CDP870, D2E7, Humira® (adalimumab), and Remicade®(infliximab); anti-TRAIL-R1 and TRAIL-R2 antibodies; anti-VE-cadherin-2antibodies; and anti-VLA-4 antibodies such as Antegren™. Furthermore,anti-idiotype antibodies including but not limited to the GD3 epitopeantibody BEC2 and the gp72 epitope antibody 105AD7, may be used. Inaddition, bispecific antibodies including but not limited to theanti-CD3/CD20 antibody Bi20 may be used.

Examples of antibodies that may be co-administered to treat autoimmuneor inflammatory disease, transplant rejection, GVHD, and the likeinclude, but are not limited to, anti-α4β7 integrin antibodies such asLDP-02, anti-beta2 integrin antibodies such as LDP-01, anti-complement(C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322,MEDI-507, anti-CD3 antibodies such as OKT3, SMART anti-CD3, anti-CD4antibodies such as IDEC-151, MDX-CD4, OKT4A, anti-CD11a antibodies,anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23antibodies such as IDEC 152, anti-CD25 antibodies such as Zenapax,anti-CD40L antibodies such as 5c8, Antova, IDEC-131, anti-CD64antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114,anti-CD147 antibodies such as ABX-CBL, anti-E-selectin antibodies suchas CDP850, anti-gpIIb/IIIa antibodies such as ReoPro/Abcixima,anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740,anti-FcγR1 antibodies such as MDX-33, anti-IgE antibodies such asrhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodiessuch as SB-240563, SCH55700, anti-IL-8 antibodies such as ABX-IL8,anti-interferon gamma antibodies, and anti-TNFa antibodies such asCDP571, CDP870, D2E7, Infliximab, MAK-195F, anti-VLA-4 antibodies suchas Antegren. Examples of other Fc-containing molecules that may beco-administered to treat autoimmune or inflammatory disease, transplantrejection, GVHD, and the like include, but are not limited to, the p75TNF receptor/Fc fusion Enbrel® (etanercept) and Regeneron's IL-1 trap.

Examples of antibodies that may be co-administered to treat infectiousdiseases include, but are not limited to, anti-anthrax antibodies suchas ABthrax, anti-CMV antibodies such as CytoGam and sevirumab,anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G,anti-helicobacter antibodies such as Pyloran, anti-hepatitis Bantibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such asHRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab,RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb,BSYX-A110, and SE-Mab.

Alternatively, the antibodies of the present invention may beco-administered or with one or more other molecules that compete forbinding to one or more Fc receptors. For example, co-administeringinhibitors of the inhibitory receptor FcγRIIb may result in increasedeffector function. Similarly, co-administering inhibitors of theactivating receptors such as FcγRIIIa may minimize unwanted effectorfunction. Fc receptor inhibitors include, but are not limited to, Fcmolecules that are engineered to act as competitive inhibitors forbinding to FcγRIIb, FcγRIIIa, or other Fc receptors, as well as otherimmunoglobulins and specifically the treatment called IVIg (intravenousimmunoglobulin). In one embodiment, the inhibitor is administered andallowed to act before the antibody is administered. An alternative wayof achieving the effect of sequential dosing would be to provide animmediate release dosage form of the Fc receptor inhibitor and then asustained release formulation of the antibody of the invention. Theimmediate release and controlled release formulations could beadministered separately or be combined into one unit dosage form.Administration of an FcγRIIb inhibitor may also be used to limitunwanted immune responses, for example anti-Factor VIII antibodyresponse following Factor VIII administration to hemophiliacs.

In one embodiment, the antibodies of the present invention areadministered with a chemotherapeutic agent. By “chemotherapeutic agent”as used herein is meant a chemical compound useful in the treatment ofcancer. Examples of chemotherapeutic agents include but are not limitedto alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan; androgenssuch as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene(Fareston); anti-metabolites such as methotrexate and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; folic acidreplenisher such as frolinic acid; nitrogen mustards such aschlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; proteins such asarginine deiminase and asparaginase; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France);topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (suchas Tomudex); additional chemotherapeutics including aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;difluoromethylornithine (DMFO); elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11;retinoic acid; esperamicins; capecitabine.Pharmaceutically acceptable salts, acids, or derivatives of any of theabove may also be used.

A chemotherapeutic or other cytotoxic agent may be administered as aprodrug. By “prodrug” as used herein is meant a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, for example Wilman, 1986, Biochemical Society Transactions, 615thMeeting Belfast, 14:375-382; Stella et al., “Prodrugs: A ChemicalApproach to Targeted Drug Delivery,” Directed Drug Delivery; andBorchardt et al., (ed.): 247-267, Humana Press, 1985, each incorporatedherein it its entirety by reference. The prodrugs that may find use withthe present invention include but are not limited tophosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, beta-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use withthe antibodies of the present invention include but are not limited toany of the aforementioned chemotherapeutic agents.

A variety of other therapeutic agents may find use for administrationwith the antibodies of the present invention. In one embodiment, theantibody is administered with an anti-angiogenic agent. By“anti-angiogenic agent” as used herein is meant a compound that blocks,or interferes to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule or aprotein, for example an antibody, Fc fusion, or cytokine, that binds toa growth factor or growth factor receptor involved in promotingangiogenesis. one such anti-angiogenic factor herein is an antibody thatbinds to Vascular Endothelial Growth Factor (VEGF). Other agents thatinhibit signaling through VEGF may also be used, for example RNA-basedtherapeutics that reduce levels of VEGF or VEGF-R expression, VEGF-toxinfusions, Regeneron's VEGF-trap, and antibodies that bind VEGF-R. In analternate embodiment, the antibody is administered with a therapeuticagent that induces or enhances adaptive immune response, for example anantibody that targets CTLA-4. Additional anti-angiogenesis agentsinclude, but are not limited to, angiostatin (plasminogen fragment),antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin, bevacizumab,bisphosphonates, BMS-275291, cartilage-derived inhibitor (CDI), CAI,CD409 complement fragment, CEP-7055, Col 3, combretastatin A-4,endostatin (collagen XVIII fragment), farnesyl transferase inhibitors,fibronectin fragment, gro-beta, halofuginone, heparinases, heparinhexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG),IM-862, interferon alpha, interferon beta, interferon gamma, interferoninducible protein 10 (IP-10), interleukin-12, kringle 5 (plasminogenfragment), marimastat, metalloproteinase inhibitors (eg. TIMPs),2-methodyestradiol, MMI 270 (CGS 27023A), plasminogen activatorinhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16 kDafragment, proliferin-related protein (PRP), PTK 7871ZK 222594,retinoids, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248,tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1(TSP-1), TNP-470, transforming growth factor beta (TGF-β),vasculostatin, vasostatin (caireticulin fragment), ZS6126,and ZD6474.

In one embodiment, the antibody is administered with a tyrosine kinaseinhibitor. By “tyrosine kinase inhibitor” as used herein is meant amolecule that inhibits to some extent tyrosine kinase activity of atyrosine kinase. Examples of such inhibitors include but are not limitedto quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline;pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo(2,3-d) pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lambert); antisensemolecules (e.g. those that bind to ErbB-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering A G);pan-ErbB inhibitors such as C1-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); Imatinib mesylate (STI571,Gleevec®; Novartis); PKI 166(Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569(Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11 (Imclone); or as described in any ofthe following patent publications: U.S. Pat. No. 5,804,396; PCT WO99/09016 (American Cyanimid); PCT WO 98/43960 (American Cyanamid); PCTWO 97/38983 (Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); PCT WO99/06396 (Warner-Lambert); PCT WO 96/30347 (Pfizer, Inc); PCT WO96/33978 (AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980(AstraZeneca), gefitinib (IRESSA™, ZD1839, AstraZeneca), and OSI-774(Tarceva™, OSI Pharmaceuticals/Genentech), each patent publicationsincorporated herein it its entirety by reference.

In another embodiment, the antibody is administered with one or moreimmunomodulatory agents. Such agents may increase or decrease productionof one or more cytokines, up- or down-regulate self-antigenpresentation, mask MHC antigens, or promote the proliferation,differentiation, migration, or activation state of one or more types ofimmune cells. Immunomodulatory agents include but are not limited to:non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin,ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin,ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib,naproxen, ketoprofen, and nabumetone; steroids (eg. glucocorticoids,dexamethasone, cortisone, hydroxycortisone, methylprednisolone,prednisone, prednisolone, trimcinolone, azulfidineicosanoids such asprostaglandins, thromboxanes, and leukotrienes; as well as topicalsteroids such as anthralin, calcipotriene, clobetasol, and tazarotene);cytokines such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine,chemokine, or receptor antagonists including antibodies, solublereceptors, and receptor-Fc fusions against BAFF, B7, CCR2, CCR5, CD2,CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28,CD40, CD40L, CD44, CD45, CD402, CD64, CD80, CD86, CD147, CD152,complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ,IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3,MHC, selectins, TGFβ, TNFα, TNFβ, TNF-R1, T-cell receptor, includingEnbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab);heterologous anti-lymphocyte globulin; other immunomodulatory moleculessuch as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypicantibodies for MHC binding peptides and MHC fragments, azathioprine,brequinar, bromocryptine, cyclophosphamide, cyclosporine A,D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold,hydroxychloroquine, leflunomide, malononitriloamides (e.g. leflunomide),methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin,and sulfasasazine.

In an alternate embodiment, antibodies of the present invention areadministered with a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormones such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

In one embodiment, cytokines or other agents that stimulate cells of theimmune system are co-administered with the antibody of the presentinvention. Such a mode of treatment may enhance desired effectorfunction. For example, agents that stimulate NK cells, including but notlimited to IL-2 may be co-administered. In another embodiment, agentsthat stimulate macrophages, including but not limited to C5a, formylpeptides such as N-formyl-methionyl-leucyl-phenylalanine(Beigier-Bompadre et al. (2003) Scand. J. Immunol. 57: 221-8,incorporated herein it its entirety by reference), may beco-administered. Also, agents that stimulate neutrophils, including butnot limited to G-CSF, GM-CSF, and the like may be administered.Furthermore, agents that promote migration of such immunostimulatorycytokines may be used. Also additional agents including but not limitedto interferon gamma, IL-3 and IL-7 may promote one or more effectorfunctions.

In an alternate embodiment, cytokines or other agents that inhibiteffector cell function are co-administered with the antibody of thepresent invention. Such a mode of treatment may limit unwanted effectorfunction.

In an additional embodiment, the antibody is administered with one ormore antibiotics, including but not limited to: aminoglycosideantibiotics (eg. apramycin, arbekacin, bambermycins, butirosin,dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,ribostamycin, sisomycin, spectrinomycin), aminocyclitols (eg.sprctinomycin), amphenicol antibiotics (eg. azidamfenicol,chloramphenicol, florfrnicol, and thiamphemicol), ansamycin antibiotics(eg. rifamide and rifampin), carbapenems (eg. imipenem, meropenem,panipenem); cephalosporins (eg. cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide,cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine),cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, andcefotetan); lincosamides (eg. clindamycin, lincomycin); macrolide (eg.azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin,tobramycin), monobactams (eg. aztreonam, carumonam, and tigernonam);mupirocin; oxacephems (eg. flomoxef, latamoxef, and moxalactam);penicillins (eg. amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, bexzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamecillin, penethamatehydriodide, penicillin o-benethamine, penicillin O, penicillin V,penicillin V benzoate, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium); polypeptides (eg. bacitracin, colistin,polymixin B, teicoplanin, vancomycin); quinolones (amifloxacin,cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine,gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin,pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin,tosufloxacin, trovafloxacin); rifampin; streptogramins (eg.quinupristin, dalfopristin); sulfonamides (sulfanilamide,sulfamethoxazole); tetracyclenes (chlortetracycline, demeclocyclinehydrochloride, demethylchlortetracycline, doxycycline, duramycin,minocycline, neomycin, oxytetracycline, streptomycin, tetracycline,vancomycin).

Anti-fungal agents such as amphotericin B, ciclopirox, clotrimazole,econazole, fluconazole, flucytosine, itraconazole, ketoconazole,niconazole, nystatin, terbinafine, terconazole, and tioconazole may alsobe used.

Antiviral agents including protease inhibitors, reverse transcriptaseinhibitors, and others, including type I interferons, viral fusioninhibitors, and neuramidase inhibitors, may also be used. Examples ofantiviral agents include, but are not limited to, acyclovir, adefovir,amantadine, amprenavir, clevadine, enfuvirtide, entecavir, foscarnet,gangcyclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin,rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, andzidovudine.

The antibodies of the present invention may be combined with othertherapeutic regimens. For example, in one embodiment, the patient to betreated with an antibody of the present invention may also receiveradiation therapy. Radiation therapy can be administered according toprotocols commonly employed in the art and known to the skilled artisan.Such therapy includes but is not limited to cesium, iridium, iodine, orcobalt radiation. The radiation therapy may be whole body irradiation,or may be directed locally to a specific site or tissue in or on thebody, such as the lung, bladder, or prostate. Typically, radiationtherapy is administered in pulses over a period of time from about 1 to2 weeks. The radiation therapy may, however, be administered over longerperiods of time. For instance, radiation therapy may be administered topatients having head and neck cancer for about 6 to about 7 weeks.Optionally, the radiation therapy may be administered as a single doseor as multiple, sequential doses. The skilled medical practitioner candetermine empirically the appropriate dose or doses of radiation therapyuseful herein. In accordance with another embodiment of the invention,the antibody of the present invention and one or more other anti-cancertherapies are employed to treat cancer cells ex vivo. It is contemplatedthat such ex vivo treatment may be useful in bone marrow transplantationand particularly, autologous bone marrow transplantation. For instance,treatment of cells or tissue(s) containing cancer cells with antibodyand one or more other anti-cancer therapies, such as described above,can be employed to deplete or substantially deplete the cancer cellsprior to transplantation in a recipient patient.

It is of course contemplated that the antibodies of the invention mayemploy in combination with still other therapeutic techniques such assurgery or phototherapy.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation.

For reference to immunoglobulin constant regions, positions are numberedaccording to the EU index as in Kabat (Kabat et al., 1991, Sequences ofProteins of Immunological Interest, 5th Ed., United States Public HealthService, National Institutes of Health, Bethesda).

Example 1 Anti-CD40 Antibodies with Amino Acid Modifications thatEnhance Effector Function

The anti-CD40 antibodies of the invention are intended as clinicalcandidates for anti-cancer therapeutics. To investigate the possibilityof improving the effector function of an antibody that targets CD40,variant versions of anti-CD40 antibodies were engineered.

FIG. 5 provides some heavy and light chain variable region sequences ofthe anti-CD40 antibodies S2C6 (Paulie S. et al., 1984. Cancer ImmunolImmunother. 17:173-179), G28-5 (Clark E. A. et al., 1988. Eur J Immunol.18:451-457), and 5D12 (de Boer M. et al., 1992. J Immunol Methods.152:15-23), each incorporated herein it its entirety by reference, usedin the present study. The mouse, parent chimeric heavy and light chainsare labeled H0 and L0, respectively. The genes for the murine WTantibody VH and VL, designated H0 and L0 respectively, were constructedusing gene synthesis techniques and subcloned into the mammalianexpression vector pcDNA3.1Zeo (Invitrogen) comprising the full lengthlight kappa (Cκ) and heavy chain IgG1 constant regions. VariantS239D/I332E (effector function enhanced anti-CD40) was constructed inthe Fc region of a hybrid IgG1/IgG2 (FIG. 1) antibody in the pcDNA3.1Zeovector using QuikChange mutagenesis techniques (Stratagene). Allsequences were sequenced to confirm the fidelity of the sequence.Plasmids containing heavy chain gene (VH-CH1-CH2-CH3) (wild-type orvariants) were co-transfected with plasmid containing light chain gene(VL-CLK) into 293T cells. Media were harvested 5 days aftertransfection, and antibodies were purified from the supernatant usingprotein A affinity chromatography (Pierce, Catalog #20334).

To assess the capacity of the antibody variants to mediate effectorfunction against CD40 expressing cells, effector function enhancedanti-CD40 antibodies were tested in a cell-based ADCC assay. Humanperipheral blood monocytes (PBMCs) were isolated from leukopaks and usedas effector cells, and CD40 positive cancer cells were used as targetcells. Target cells were seeded at 20,000 cells/well and treated withdesignated antibodies in triplicates. PBMCs isolated using a Ficollgradient were added in excess to target cells and co-cultured for 4 hrsbefore processing for LDH activity using the Cytotoxicity Detection Kitaccording to the manufacturer's instructions. FIG. 6 shows the resultsof ADCC assays comparing WT IgG1, effector function enhanced anti-CD40antibodies (hybrid S239D/I332E), and/or effector function enhancedrituximab (hybrid S239D/I332E) on the cell lines Daudi (Burkitt'sLymphoma), Raji (Burkitt's Lymphoma), and RPMI8226 (Multiple Myeloma)for antibodies S2C6 (FIG. 6 a), 5D12 (FIG. 6 b), and G28-5 (FIG. 6 c).The graphs show that the antibodies differ not only in their EC50,reflecting their relative potency, but also in the maximal level of ADCCattainable by the antibodies at saturating concentrations, reflectingtheir relative efficacy. These two terms, potency and efficacy, aresometimes used loosely to refer to desired clinical properties. In thecurrent experimental context, however, they are denoted as specificquantities, and therefore are here explicitly defined. By “potency” asused in the current experimental context is meant the EC50 of anantibody. By “efficacy” as used in the current experimental context ismeant the maximal possible effector function of an antibody atsaturating levels. Considerable enhancements in potency and efficacy areobserved for the Fc variant antibodies with enhanced effector functionas compared to the antibodies with WT Fc region. All three effectorfunction enhanced antibodies displayed ADCC on the Multiple Myeloma cellline RPMI8226, with S2C6 showing the most efficacy, indicating that thisantibody may be an effective treatment option for Multiple Myeloma.

Example 2 Platelet Activation Assay with Anti-CD40 Antibody S2C6

To determine if anti-CD40 antibody S2C6 has any effect of plateletactivation, a platelet activation assay was performed. Platelets wereobtained from fresh blood drawn by venipuncture in NaCitrate and spun atlow speed (100×g) for 15 minutes from which the straw colored upperphase was removed as platelet rich plasma (PRP). The PRP was furtherspun at 700×g for 2.5 minutes and the pellet was saved and washed twicewith 10 mM EDTA, 1% FBS in CaCl₂ and MgCl₂ free PBS. The platelet pelletwas resuspended in EDTA/FBS/PBS at 1.5× of the PRP volume and used inthe experiment. Antibody serial dilutions were performed in EDTA/FBS/PBSin 50 μl volume in 96 well microtiter plates with 5× molar access ofcross linking antibody. For IV.3 antibody (mouse anti-human IgG FcgRIIaspecific antibody), goat anti-mouse IgG Fc specific antibody was usedfor cross-linking and for all other antibodies goat anti-human IgG Fcspecific biotinylated antibody was used for cross-linking. To theantibody dilution wells, 50 μL of prepared washed platelets were added.Samples were incubated for 35 minutes at 37° C. and developed with theATP dependent luminescence without SDS kit (ATP-Lite, PerkinElmer).Results for the platelet activation assay are shown in FIG. 7. Theresults show that neither S2C6 IgG1 or effector function enhanced S2C6activated platelets at concentrations of less than 10000 ng/mL, whilethe positive control antibody IV.3 clearly mediates platelet activationat all concentrations.

Example 3 Anti-Proliferation Assay of Anti-CD40 Antibodies on theBurkitt's Lymphoma Cell Line HS-Sultan

To observe an anti-proliferative effect in vitro, many antibodiesrequire cross-linking, usually accomplished by a secondary antibody. Ithas been proposed that corresponding in vivo effects for theseantibodies may be dependent on cross-linking mediated by Fc receptorsexpressed on the surface of effector cells. In this experiment,anti-proliferative effects of the chimeric antibodies S2C6 and 5D12 wereassayed using the Burkitt's Lymphoma cell line HS-Sultan (FIG. 8). TheHS-Sultan cells were maintained in 10% FBS/RPMI1640 with NaPyruvate andHEPES. The antibody serial dilutions were prepared in 96 well microtiterplates with 10× molar access of cross-linking antibodies in 50 μl of 2%FBS/RPMI1640 with HEPES and NaPyruvate. In some cases, additionalfactors are added at a fixed concentration as indicated. Thecross-linking antibodies used were goat anti-human IgG Fc specificbiotinylated antibody and goat anti-mouse IgG Fc specific antibody forhuman or mouse antibodies, respectively. HS-Sultan cells were pelletedand resuspended in 2% FBS/RPMI1640 with HEPES and NaPyruvate at 100,000cells/ml and 50 μl was added to the prepared antibody dilutions. After 4days at 37° C., the amount of ATP present in the live cells was detectedusing the ATP dependent luminescent viability assay kit (Cell TiterGlo,Promega Corp.). Human IgG with and without CD40 ligand and cells onlywere used as controls. Both S2C6 and 5D12 show anti-proliferativeeffects on the HS-Sultan cell line.

Example 4 Anti-CD40 Antibodies with Reduced Potential for Immunogenicity

In order to reduce the potential for immunogenicity of the H0 and L0S2C6 variable regions, the immunogenicity was reduced using a methoddescribed in U.S. Ser. No. 11/004,590, entitled “Methods of GeneratingVariant Proteins with Increased Host String Content and CompositionsThereof”, filed on Dec. 6, 2004. The method reduces the potential forimmunogenicity by increasing the human string content of the antibodythrough mutations. The anti-CD40 variable region variants with reducedpotential for immunogenicity are named H1, H2, H3, and H4 for the heavychain, and L1, L2, and L3 for the light chain. The sequences for thesevariants are provided in FIG. 9. Light and heavy chains were constructedby gene synthesis. Light chains were subcloned into the pcDNA3.1Zeovector comprising the full length light kappa (GO constant region, andheavy chains were subcloned into pcNDA3.1Zeo vectors comprising both theWT IgG1 constant chain (designated IgG1_WT), as well as the heavy chainHybrid IgG constant region with mutations S239D/I332E (designatedHybrid_S239D/I332E). All sequences were sequenced to confirm thefidelity of the sequence. Combinations of the different heavy and lightchains were expressed in 293T cells and purified as described above. Forexample, an S2C6 antibody comprising the H3 heavy chain and L1 lightchain would be designated as S2C6_H3L1.

ADCC was used to differentiate the reduced immunogenicity templates.Templates were incubated at 60° C. for up to 48 hrs and ADCC measured onRAMOS cells at various time points in order to assess the relativestability of each template (FIG. 11). Human peripheral blood monocytes(PBMCs) were isolated from leukopaks and used as effector cells, andCD40 positive cancer cells were used as target cells. Target cells wereseeded in 96-well plates and treated with designated antibodies intriplicate. PBMCs isolated using a Ficoll gradient were added in excessto target cells and co-cultured for 4 hrs before processing for LDHactivity using the Cytotoxicity Detection Kit according to themanufacturer's instructions. After a 48 hr incubation, reducedimmunogenicity templates H1L1, H1L3, H2L1, and H2L3 still maintainedacceptable ADCC potency and efficacy. Based on ADCC potency and relativestability, a reduced immunogenicity (humanized) S2C6 antibody was chosenfor further development.

Example 5 ADCC of Reduced Immunogenicity S2C6 on Ramos, Namalwa, IM-9,and RPMI Cells

In order to evaluate cytotoxic properties of effector function enhancedand reduced immunogenicity S2C6 we performed ADCC assays on the celllines Ramos (Burkitt's Lymphoma), Namalwa (Burkitt's Lymphoma), IM-9(B-Iymphoblastoid), and RPMI8226 (Multiple Myeloma) (FIGS. 12 a-12 d,respectively). For Ramos and Namalwa cell lines, human peripheral bloodmonocytes (PBMCs) were isolated from leukopaks and used as effectorcells, and the CD40 positive cancer cells were used as target cells.Target cells were seeded at 10,000 cells/well in 96-well plates andtreated with designated antibodies in triplicate. PBMCs isolated using aFicoll gradient were added in excess to target cells and co-cultured for4 hrs before processing for LDH activity using the CytotoxicityDetection Kit according to the manufacturer's instructions. For IM-9 andRPMI8226, ADCC assays were performed with purified NK cells. ADCC withpurified NK cells is done in 96-well microtiter plates. The NK cellswere purified from human PBMC using the kit from Miltenyi Biotec (Cat#130-091-152) and incubated in 10% FBS/RPMI1640 overnight with 10 ng/mlIL-2. The following day, 10,000 cancer target cells are opsonized withvarying concentrations of antibody and 50k NK cells are used for eachantibody concentration in triplicates. The target cells are washed threetimes while NK cells are washed twice with RPMI1640 and both resuspendedin 1% FBS/RPMI1640 and added to the antibody solutions. After 4 hours ofincubation at 37° C. in a humidified incubator with 5% CO₂, the assaywas quantified using LDH dependent CytoTox-One fluorescence dependentdetection system from Promega (#PAG7891). Total LDH signal is determinedfrom the Triton-X100 lysed target cells (Total Target LDH) and used tonormalize against the spontaneous LDH background (SpontaneousBackground) adjusted experimental values. Thus % ADCC=((ExperimentalValue−Spontaneous Background)/(Total Target LDH−Target LDH))*100.Spontaneous background is the value obtained from the Target and NKcells co-incubated in the absence of antibody. Target LDH is the valuefrom the target cancer cells alone spontaneously releasing LDH duringthe incubation. As can be seen in FIG. 12 a, S2C6 H3L1 Hybrid S239D/I332mediates very high levels of ADCC on the Burkitt's Lymphoma cell lineRamos. In FIG. 12 b, S2C6 H4L3 Hybrid S239D/I332E mediates a high levelsof ADCC on the Burkitt's Lymphoma cell line Namalwa. In FIG. 12 c, S2C6H1L1 Hybrid S239D/I332E mediates high levels of ADCC on theB-lymphoblastoid cell line IM-9. Finally, in FIG. 12 d, S2C6 H1L1 HybridS239D/I332E mediates high levels of ADCC on the multiple myeloma cellline RPMI8226. Rituximab and WT IgG1 antibodies are also used in some ofthe assays for comparison. These assays show that humanized and effectorfunction enhanced S2C6 has potent in vitro efficacy on several differentcell lines representing B-cell malignancies.

Example 6 ADCC of an Enhanced Effector Function Anti-CD40 Antibody withReduced Fucose Content

Anti-CD40 antibodies with enhanced effector function (e.g., S2C6 H1 L1Hybrid S239D/I332E) are evaluated with reduced fucose content. The Lec13cell line (Ripka et al. Arch. Biochem. Biophys. 49:533-545 (1986)) isutilized to express anti-CD40 antibodies with reduced fucose content.Lec13 refers to the lectin-resistant Chinese Hamster Ovary (CHO) mutantcell line which displays a defective fucose metabolism and therefore hasa diminished ability to add fucose to complex carbohydrates. That cellline is described in Ripka & Stanley, 1986, Somatic Cell & Molec. Gen.12(1):51-62; and Ripka et al., 1986, Arch. Biochem. Biophys.249(2):533-545. Lec13 cells are believed to lack the transcript forGDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism.Ohyama et al., 1988, J. Biol. Chem. 273(23):14582-14587.GDP-D-mannose-4,6-dehydratase generatesGDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is thenconverted by the FX protein to GDP-L-fucose. Expression of fucosylatedoligosaccharides is dependent on the GDP-L-fucose donor substrates andfucosyltransferase(s). The Lec13 CHO cell line is deficient in itsability to add fucose, but provides IgG with oligosaccharide which isotherwise similar to that found in normal CHO cell lines and from humanserum (Jefferis, R. et al., 1990, Biochem. J. 268, 529-537; Raju, S. etal., 2000, Glycobiology 10, 477-486; Routier, F. H., et al., 1997,Glycoconj. J. 14, 201-207). Normal CHO and HEK293 cells add fucose toIgG oligosaccharide to a high degree, typically from 80-98%, and IgGsfrom sera are also highly fucosylated (Jefferis, R. et al., 1990,Biochem. J. 268, 529-537; Raju, S. et al., 2000, Glycobiology 10,477-486; Routier, F. H., et al., 1997, Glycoconj. J. 14, 201-207;Shields et al., 2002, J Biol Chem 277(90):26733-26740). It is wellestablished that antibodies expressed in transfected Lec13 cellsconsistently produce about 10% fucosylated carbohydrate (Shields et al.,2002, J Biol Chem 277(90):26733-26740).

ADCC assays are performed with purified NK cells on Raji and RPMI8226cells using anti-CD40 antibodies with and without enhanced effectorfunction variants and with and without reduced fucosylation. It isexpected that both ADCC potency and efficacy for anti-CD40 antibody withreduced fucose content (S2C6_H1L1_(—)1gG1_WT-fucose) are significantlyhigher than those of the antibody with native fucose content(S2C6_H1L1_IgG1_WT) and similar to those of the antibody with amino acidmodification (S2C6_H1L1_Hybrid_(—)239D/I332E+fucose). This experimentwill illustrate that combinations of amino acid modifications andmodified glycoforms may be used to optimize anti-CD40 antibodies foreffector function properties.

The use of the Lec13 cell line is not meant to limit the presentinvention to that particular mode of reducing fucose content. A varietyof other methods are known in the art for controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, including but not limited to expression invarious organisms or cell lines, engineered or otherwise (for exampleLec13 CHO cells or rat hybridoma YB2/0 cells), regulation of enzymesinvolved in the glycosylation pathway (for example FUT8[α1,6-fucosyltranserase] and/or β1-4-N-acetylglucosaminyltransferase III[GnTIII]), and modification of modifying carbohydrate(s) after the IgGhas been expressed (Umaña et al., 1999, Nat Biotechnol 17:176-180;Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002,J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem278:3466-3473; Yamane-Ohnuki et al., 2004, Biotechnology andBioengineering 87(5):614-621); (U.S. Pat. No. 6,602,684; U.S. Ser. No.10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1).

Example 7 Human Tumor Xenograft Models

In the first study, five to six week old SCID mice were injectedsubcutaneously (s.c.) with 5×10⁶ human Ramos Burkitt's lymphoma cells.On day 14, mice bearing tumors of 40-100 mm³ were injectedintraperitoneally (i.p.) with 6 mg/kg of S2C6 H1L1 Hybrid S239D/I332E,isotype control (Hybrid S239D/I332E Fc), or with PBS, 3× per week for 3weeks. Tumors were measured twice per week and tumor volume wasdetermined using the formula L×W²/2, where L=length and W=width. Tumorgrowth was monitored for 40 days post tumor cell injection.

In a separate study, five to six week old SCID mice were injectedintravenously (i.v.) with 5×10⁶ human Ramos Burkitt's lymphoma cells. Ondays 3, 6, 10, 13 and 17 post tumor cell injection, mice were injectedintraperitoneally (i.p.) with 6, 2 or 0.6 mg/kg of S2C6 H1L1 HybridS239D/I332E, isotype control (Hybrid S239D/I332E Fc), or with PBS. Micewere monitored for survival for 60 days. As shown in FIG. 13, S2C6 H1 L1Hybrid S239/I332E significantly prolonged the survival of these animals.

The use of particular modifications, for example the substitutions 239Dand 332E, to enhance effector function are not meant to constrain theanti-CD40 antibodies to these particular modifications. As describedabove in the section entitled “Modifications for optimizing effectorfunction”, a large number of modifications, including amino acidmodifications and modified glycoforms, are contemplated for anti-CD40antibodies to improve their effector function properties.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

What is claimed is:
 1. An antibody that binds CD40, said antibodycomprising a heavy chain and/or a light chain, wherein said heavy chaincomprises a V_(H) domain having an amino acid sequence selected from thegroup consisting of the amino acid sequence of SEQ ID NOs:34-37, andwherein said light chain comprises a V_(L) domain having an amino acidsequence selected from the group consisting of SEQ ID NOs: 38 and
 40. 2.The antibody of claim 1, wherein said heavy chain comprises a V_(H)domain having an amino acid sequence selected from the group consistingof the amino acid sequence of SEQ ID NOs:34-35, and wherein said lightchain comprises a V_(L) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 38 and
 40. 3. The antibody ofclaim 2 further comprising a modification in the constant regionrelative to the parent anti-CD40 antibody, wherein said modification isan amino acid substitution selected from the group consisting of 236A,239D, 268D, 330Y, 330L, and 332E, and wherein the numbering system is asin Kabat, and wherein said antibody binds with increased affinity to theFcyRIIIa receptor as compared to the parent antibody.
 4. The antibody ofclaim 3, wherein said antibody comprises a heavy chain V_(H) domainhaving an amino acid sequence of SEQ ID NO:34 and a light chain V_(L)domain having an amino acid sequence of SEQ ID NO:38.
 5. A variantanti-CD40 antibody variable domain relative to antibody S2C6 variabledomain comprising a heavy chain, SEQ ID NO:10, and a light chain, SEQ IDNO:11, wherein said variant comprises at least one set selected from thegroup consisting of: a first set, a second set, a third set, a fourthset, a fifth set, and a sixth set, wherein said first set consists ofsubstitutions Q5V, P9A, D10E, L11V and V12K relative to SEQ ID NO:10,wherein said second set consists of substitutions K38R, S40A, and H41Prelative to SEQ ID NO:10, wherein said third set consists ofsubstitutions K67R, A68V, I69T, L70I, and T71S relative to SEQ ID NO:10,wherein said fourth set consists of substitutions T109L and L110Vrelative to SEQ ID NO:10, wherein said fifth set consists ofsubstitutions T7S, A17E and Q18P relative to SEQ ID NO:11, and, whereinsaid sixth set consists of substitutions K50Q, L88V and Q112K relativeto SEQ ID NO:11.
 6. A method of treating a B-cell related diseaseselected from the group consisting of: non-Hodgkin's lymphomas (NHL),chronic lymphocytic leukemia (CLL), B-cell acute lymphoblasticleukemia/lymphoma (B-ALL), mantle cell lymphoma (MCL), hairy cellleukemia (HCL), chronic myelogenous leukemia (CML), and multiple myeloma(MM), wherein said method comprises administering an antibody accordingto claim
 1. 7. A method of treating a human solid tumor expressing CD40,wherein said method comprises administering an antibody according toclaim
 1. 8. The method of claim 6 or 7, wherein said antibody isadministered in combination with at least one agent selected from thegroup consisting of a cytotoxic agent, a chemotherapeutic agent, acytokine, a growth inhibitory agent, an anti-hormonal agent, a kinaseinhibitor, an anti-angiogenic agent, a cardioprotectant, animmunostimulatory agent, an immunosuppressive agent, an agent thatpromotes proliferation of hematological cells, an angiogenesisinhibitor, a protein tyrosine kinase inhibitor, and a second antibody.9. A composition comprising an antibody according to claim 1 and anacceptable carrier.
 10. A method of inhibiting proliferation of a cellexpressing CD40, wherein said method comprises contacting said cell withan antibody according to claim
 1. 11. A method of enhancing antibodydependent cell cytotoxicity toward a cell expressing CD40, wherein saidmethod comprises contacting said cell with an antibody according toclaim
 1. 12. A method of depleting a mammal of at least one cellexpressing CD40, wherein said method comprises administering to themammal an antibody according to claim
 1. 13. The antibody of claim 3,wherein said antibody comprises a heavy chain V_(H) domain having anamino acid sequence of SEQ ID NO:34 and a light chain V_(L) domainhaving an amino acid sequence of SEQ ID NO:40.
 14. A method of treatinga B-cell related disease selected from the group consisting of:non-Hodgkin's lymphomas (NHL), chronic lymphocytic leukemia (CLL),B-cell acute lymphoblastic leukemia/lymphoma (B-ALL), mantle celllymphoma (MCL), hairy cell leukemia (HCL), chronic myelogenous leukemia(CML), and multiple myeloma (MM), wherein said method comprisesadministering an antibody according to claim
 13. 15. A method oftreating a human solid tumor expressing CD40, wherein said methodcomprises administering an antibody according to claim
 13. 16. Themethod of claim 14, wherein said antibody is administered in combinationwith at least one agent selected from the group consisting of acytotoxic agent, a chemotherapeutic agent, a cytokine, a growthinhibitory agent, an anti-hormonal agent, a kinase inhibitor, ananti-angiogenic agent, a cardioprotectant, an immunostimulatory agent,an immunosuppressive agent, an agent that promotes proliferation ofhematological cells, an angiogenesis inhibitor, a protein tyrosinekinase inhibitor, and a second antibody.
 17. The method of claim 15,wherein said antibody is administered in combination with at least oneagent selected from the group consisting of a cytotoxic agent, achemotherapeutic agent, a cytokine, a growth inhibitory agent, ananti-hormonal agent, a kinase inhibitor, an anti-angiogenic agent, acardioprotectant, an immunostimulatory agent, an immunosuppressiveagent, an agent that promotes proliferation of hematological cells, anangiogenesis inhibitor, a protein tyrosine kinase inhibitor, and asecond antibody.
 18. A composition comprising an antibody according toclaim 13 and an acceptable carrier.
 19. A method of inhibitingproliferation of a cell expressing CD40, wherein said method comprisescontacting said cell with an antibody according to claim
 13. 20. Amethod of enhancing antibody dependent cell cytotoxicity toward a cellexpressing CD40, wherein said method comprises contacting said cell withan antibody according to claim
 13. 21. A method of depleting a mammal ofat least one cell expressing CD40, wherein said method comprisesadministering to the mammal an antibody according to claim
 13. 22. Amethod of inhibiting the proliferation of a cell expressing CD40 in apatient having an autoimmune disease selected from the group consistingof allogenic islet graft rejection, alopecia greata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases ofthe adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritisand orehitis, autoimmune thrombocytopenia, autoimmune urticaria,Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman'ssyndrome, celiac spruce-dermatitis, chronic fatigue immune dysfunctionsyndrome, chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, dermatomyositis, discoid lupus,essential mixed cryoglobulinemia, factor VIII deficiency,fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease,Guillain-Barre, Goodpasture's syndrome, graft-versus-host disease(GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, IgMpolyneuropathies, immune mediated thrombocytopenia, juvenile arthritis,Kawasaki's disease, lichen plantus, lupus erythematosus, Meniere'sdisease, mixed connective tissue disease, multiple sclerosis, type 1diabetes mellitus, myasthenia gravis, pemphigus vulgaris, perniciousanemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobinulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Reynauld's phenomenon, Reiter's syndrome, rheumatoidarthritis, sarcoidosis, scleroderma, Sjorgen's syndrome, solid organtransplant rejection, stiff man syndrome, systemic lupus erythematosus,takayasu arteritis, temporal arteristis/giant cell arteritis, thromboticthrombocytopenia purpura, ulcerative colitis, uveitis, vasculitides suchas dermatitis herpetiformis vasculitis, vitiligo, and Wegner'sgranulomatosis, the method comprising administering the antibody ofclaim
 1. 23. A method of inhibiting the proliferation of a cellexpressing CD40 in a patient having an autoimmune disease selected fromthe group consisting of allogenic islet graft rejection, alopeciagreata, ankylosing spondylitis, antiphospholipid syndrome, autoimmuneAddison's disease, antineutrophil cytoplasmic autoantibodies (ANCA),autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia,autoimmune oophoritis and orehitis, autoimmune thrombocytopenia,autoimmune urticaria, Behcet's disease, bullous pemphigoid,cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronicfatigue immune dysfunction syndrome, chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, dermatomyositis,discoid lupus, essential mixed cryoglobulinemia, factor VIII deficiency,fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease,Guillain-Barre, Goodpasture's syndrome, graft-versus-host disease(GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, IgMpolyneuropathies, immune mediated thrombocytopenia, juvenile arthritis,Kawasaki's disease, lichen plantus, lupus erythematosus, Meniere'sdisease, mixed connective tissue disease, multiple sclerosis, type 1diabetes mellitus, myasthenia gravis, pemphigus vulgaris, perniciousanemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobinulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Reynauld's phenomenon, Reiter's syndrome, rheumatoidarthritis, sarcoidosis, scleroderma, Sjorgen's syndrome, solid organtransplant rejection, stiff man syndrome, systemic lupus erythematosus,takayasu arteritis, temporal arteristis/giant cell arteritis, thromboticthrombocytopenia purpura, ulcerative colitis, uveitis, vasculitides suchas dermatitis herpetiformis vasculitis, vitiligo, and Wegner'sgranulomatosis, the method comprising administering an antibodyaccording to claim
 13. 24. A method of inhibiting the proliferation of acell expressing CD40 in a patient having a cancer selected from thegroup consisting of Burkitt's lymphoma (BL), small lymphocyticlymphoma/chronic lymphocytic leukemia (SLL/CLL), mantle cell lymphoma(MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLCL),marginal zone lymphoma (MZL), hairy cell leukemia (HCL)lymphoplasmacytic leukemia (LPL), extranodal marginal zone B-celllymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginalzone B cell lymphoma, mediastinal large cell lymphoma, intravascularlarge cell lymphoma, primary effusion lymphoma, precursorB-lymphoblastic leukemia/lymphoma, blastic NK cell lymphoma, largegranular lymphocytic leukemia, aggressive NK-cell leukemia, extranodalNK cell lymphoma, anaplastic large cell lymphoma (ALCL), mycosisfungoides/Sezary syndrome, Hodgkin's lymphoma, B-cell acutelymphoblastic leukemia/lymphoma (B-ALL), Langerhans cell histocytosis,multiple myeloma (MM), myelogenous leukemia, acute myelogenous leukemia(AML), AML with maturation, AML without differentiation, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, myelodysplastic syndrome, chronic myeloproliferative disorder(MDS), and chronic myelogenous leukemia (CML), the method comprisingadministering an antibody according to claim
 1. 25. A method ofinhibiting the proliferation of a cell expressing CD40 in a patienthaving a cancer selected from the group consisting of Burkitt's lymphoma(BL), small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL),mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse largeB-cell lymphoma (DLCL), marginal zone lymphoma (MZL), hairy cellleukemia (HCL) lymphoplasmacytic leukemia (LPL), extranodal marginalzone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodalmarginal zone B cell lymphoma, mediastinal large cell lymphoma,intravascular large cell lymphoma, primary effusion lymphoma, precursorB-lymphoblastic leukemia/lymphoma, blastic NK cell lymphoma, largegranular lymphocytic leukemia, aggressive NK-cell leukemia, extranodalNK cell lymphoma, anaplastic large cell lymphoma (ALCL), mycosisfungoides/Sezary syndrome, Hodgkin's lymphoma, B-cell acutelymphoblastic leukemia/lymphoma (B-ALL), Langerhans cell histocytosis,multiple myeloma (MM), myelogenous leukemia, acute myelogenous leukemia(AML), AML with maturation, AML without differentiation, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, myelodysplastic syndrome, chronic myeloproliferative disorder(MDS), and chronic myelogenous leukemia (CML), the method comprisingadministering an antibody according to claim
 13. 26. A method ofinhibiting the proliferation of a cell expressing CD40 in a patienthaving a cancer selected from the group consisting of glioma,glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma,retinoblastoma, nasopharyngeal cancer, salivary gland carcinoma,esophageal cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung,gastrointestinal cancer, cancer of the bile duct, cancer of the biliarytract, colon cancer, rectal cancer, colorectal cancer, anal carcinoma,testicular cancer, penile cancer, prostate cancer, uterine cancer,vaginal cancer, vulval cancer, cervical cancer, ovarian cancer,endometrial cancer, melanoma, basal cell carcinoma, squamous cellcancer, actinic keratosis, liver cancer, hepatic carcinoma,hepatocellular cancer, hepatoma, osteoclastoma, osteolytic bone cancer,pancreatic cancer, bladder cancer, kidney cancer, renal cancer, thyroidcancer, breast cancer, cancer of the peritoneum, Kaposi's sarcoma,angiosarcoma, and hemagiopericytoma, the method comprising administeringan antibody according to claim
 1. 27. A method of inhibiting theproliferation of a cell expressing CD40 in a patient having a cancerselected from the group consisting of glioma, glioblastoma,neuroblastoma, astrocytoma, medulloblastoma, ependymoma, retinoblastoma,nasopharyngeal cancer, salivary gland carcinoma, esophageal cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, gastrointestinal cancer,cancer of the bile duct, cancer of the biliary tract, colon cancer,rectal cancer, colorectal cancer, anal carcinoma, testicular cancer,penile cancer, prostate cancer, uterine cancer, vaginal cancer, vulvalcancer, cervical cancer, ovarian cancer, endometrial cancer, melanoma,basal cell carcinoma, squamous cell cancer, actinic keratosis, livercancer, hepatic carcinoma, hepatocellular cancer, hepatoma,osteoclastoma, osteolytic bone cancer, pancreatic cancer, bladdercancer, kidney cancer, renal cancer, thyroid cancer, breast cancer,cancer of the peritoneum, Kaposi's sarcoma, angiosarcoma, andhemagiopericytoma, the method comprising administering an antibodyaccording to claim 13.